CIDE-B polypeptides

ABSTRACT

The present invention relates to a purified or isolated polynucleotide encoding human CIDE B protein, the regulatory nucleic acids contained therein, polymorphic markers thereof, and the resulting encoded protein, as well as to methods and kits for detecting this polynucleotide and this protein. The present invention also pertains to a polynucleotide carrying the natural regulatory regions of the CIDE B gene which is useful, for example, to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory regions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.09/807,166, filed Sep. 10, 2001, now U.S. Pat. No. 6,472,517 which is anational stage of PCT/IB99/01702, filed Oct. 8, 1999, which claims thebenefit of U.S. provisional application Ser. No. 60/103,729, filed Oct.9, 1998.

FIELD OF THE INVENTION

The present invention relates to a purified or isolated polynucleotideencoding human CIDE B protein, the regulatory nucleic acids containedtherein, polymorphic markers thereof, and the resulting encoded protein,as well as to methods and kits for detecting this polynucleotide andthis protein. The present invention also pertains to a polynucleotidecarrying the natural regulatory regions of the CIDE B gene which isuseful, for example, to express a heterologous nucleic acid in hostcells or host organisms as well as functionally active regulatorypolynucleotides derived from said regulatory regions.

BACKGROUND OF THE INVENTION

Apoptosis is of fundamental importance to biological processes includingembryogenesis, maintenance of tissue homeostasis, normal cellulardevelopment of multicellular organisms, elimination of virus-infectedcells, and the development of immune system. It is a type of death thatis fundamentally distinct from degenerative death or necrosis in that itis an active process of gene-directed cellular self-destruction which,in some instances, serves a biologically meaningful homeostaticfunction. Necrosis, in contrast, is cell death occurring as a result ofsevere injurious changes in the environment of infected cells.

Morphologically, apoptosis is characterized by the rapid condensation ofthe cell with preservation of membranes. Synchronistically with thecompaction of chromatin, several biochemical changes occur in the cell.Nuclear DNA is cleaved at the linker regions between nucleosomes toproduce fragments that are easily demonstrated by agarose gelelectrophoresis wherein a characteristic ladder develops.

The primary image of apoptosis is that of the dying thymocyte: fusion ofchromatin into one mass, which binds to the nuclear membrane, while thecytoplasm remains apparently intact before beginning to condense. Thenuclear change is one of the earliest visible processes; the conversionto the condensed state occurs rapidly and is accompanied byendonucleolytic degradation of DNA between nucleosomes. Once thechromatin has condensed, electrophoresis of the DNA demonstrates aladder of fragments differing in size by 180 bp, generated by anenzymatic activity resembling that of DNase I.

This type of cell death is seen in many varieties of cells, especiallythose that, like lymphocytes or thymocytes, have relatively littlecytoplasm and are highly mitotic or derive from highly mitotic lines. Inthis situation, in which mitotic cells are likely to face challenges bymutagens (viruses, toxins), an appropriate biological imperative wouldbe to destroy the DNA rapidly and effectively. Thus, this type of celldeath is particularly dramatic among hematopoietic cells and theirderivatives.

Several regulatory components of the apoptotic pathway have beenidentified in various living organisms including man and the nematodeCaenorhabditis elegans.

Two murine transcription products involved in cell apoptosis have beenreported by Inohara et al. (1998), that have been named respectivelyCIDE-A and CIDE-B. Murine CIDE-A and CIDE-B have strong homology withthe murine anti-apoptosis DFF45 protein as well as with the drosophilaprotein DREP-1. The homology of CIDE-A, CIDE-B and FSP27 with DFF45 wasrestricted to an N-terminal region designated by Inohara et al. asCIDE-N domain which showed 39, 29 and 38% amino acid identityrespectively with DFF45.

Because there is a strong need in the art to make available to thepublic novel means useful to prevent or inhibit apoptosis disorders,either in the case of disorders caused by abnormal cell proliferationwherein apoptosis induction is desirable or in the case of disorderscaused by abnormal cell death wherein an inhibition or an arrest ofapoptosis is desirable, the inventors have attempted to isolate andcharacterize a novel gene encoding a protein involved in apoptosispathway, namely the human CIDE-B gene.

SUMMARY OF THE INVENTION

The present invention pertains to a nucleic acid molecule comprising thegenomic sequence of the human CIDE B gene. The CIDE B genomic sequencecomprises regulatory sequences located both upstream (5′-end) anddownstream (3′-end) of the transcribed portion of said gene, theseregulatory sequences being also part of the invention.

The invention also deals with the complete cDNA sequence encoding theCIDE B protein, as well as with the corresponding translation product.

Oligonucleotide probes or primers hybridizing specifically with a CIDE Bgenomic or cDNA sequence are also part of the present invention.

A further object of the invention consists of recombinant vectorscomprising any of the nucleic acid sequences above described, and inparticular of recombinant vectors comprising a CIDE B regulatorysequence or a sequence encoding a CIDE B protein, as well as of cellhosts comprising said nucleic acid sequences or recombinant vectors.

Finally, the invention is directed to methods for the screening ofsubstances or molecules which modulate the expression of CIDE B.

The invention is also directed to biallelic markers that are locatedwithin the CIDE B genomic sequence, these biallelic markers representinguseful tools in order to identify a statistically significantassociation between specific alleles of CIDE B gene and one or severaldisorders related to apoptosis such as cancer and AIDS.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagram of the CIDE B1 gene with an indication of therelative position of the biallelic markers of the present invention. Theupper line refers to the genomic sequence of CIDE B. The middle linerefers to the cDNA. The lower line refers to the CIDE B protein.

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

SEQ ID No 1 contains a genomic sequence of CIDE B comprising the 5′regulatory region (upstream untranscribed region), the exons andintrons, and the 3′ regulatory region (downstream untranscribed region).

SEQ ID No 2 contains a cDNA sequence of CIDE B.

SEQ ID No 3 contains the amino acid sequence encoded by the cDNA of SEQID No 2.

SEQ ID No 4 contains a primer containing the additional PU 5′ sequencedescribed further in Example 2

SEQ ID No 5 contains a primer containing the additional RP 5′ sequencedescribed further in Example 2.

In accordance with the regulations relating to Sequence Listings, thefollowing codes have been used in the Sequence Listing to indicate thelocations of biallelic markers within the sequences and to identify eachof the alleles present at the polymorphic base. The code “r” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is an adenine. The code “y” in thesequences indicates that one allele of the polymorphic base is athymine, while the other allele is a cytosine. The code “m” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is an cytosine. The code “k” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a thymine. The code “s” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a cytosine. The code “w” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is an thymine. The nucleotide code ofthe original allele for each biallelic marker is the following:

Biallelic marker Original allele 12-73-49 C 12-74-38 T

DETAILED DESCRIPTION OF THE INVENTION

The aim of the present invention is to provide the human CIDE B gene,the human CIDE B mRNA molecules and the polynucleotides derived fromthem. The polynucleotides of the invention are useful to design suitablemeans for detecting the presence of this gene or cDNA in a test sampleand to design suitable means to express a desired polynucleotide ofinterest. The invention also relates to the human CIDE B polypeptide.

Definitions

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

The terms “CIDE B gene”, when used herein, encompasses genomic, mRNA andcDNA sequences encoding the CIDE B protein.

The term “heterologous protein”, when used herein, is intended todesignate any protein or polypeptide other than the CIDE B protein. Moreparticularly, the heterologous protein is a compound which can be usedas a marker in further experiments with a CIDE B regulatory region.

The term “isolated” requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

The term “purified” does not require absolute purity; rather, it isintended as a relative definition. Purification of starting material ornatural material to at least one order of magnitude, preferably two orthree orders, and more preferably four or five orders of magnitude isexpressly contemplated. As an example, purification from 0.1%concentration to 10% concentration is two orders of magnitude. The term“purified” is used herein to describe a polynucleotide or polynucleotidevector of the invention which has been separated from other compoundsincluding, but not limited to other nucleic acids, carbohydrates, lipidsand proteins (such as the enzymes used in the synthesis of thepolynucleotide), or the separation of covalently closed polynucleotidesfrom linear polynucleotides. A polynucleotide is substantially pure whenat least about 50%, preferably 60 to 75% of a sample exhibits a singlepolynucleotide sequence and conformation (linear versus covalentlyclose). A substantially pure polynucleotide typically comprises about50%, preferably 60 to 90% weight/weight of a nucleic acid sample, moreusually about 95%, and preferably is over about 99% pure. Polynucleotidepurity or homogeneity is indicated by a number of means well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polynucleotide band uponstaining the gel. For certain purposes higher resolution can be providedby using HPLC or other means well known in the art.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides, for example, polypeptides which include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

The term “purified” is used herein to describe a polypeptide of theinvention which has been separated from other compounds including, butnot limited to nucleic acids, lipids, carbohydrates and other proteins.A polypeptide is substantially pure when at least about 50%, preferably60 to 75% of a sample exhibits a single polypeptide sequence. Asubstantially pure polypeptide typically comprises about 50%, preferably60 to 90% weight/weight of a protein sample, more usually about 95%, andpreferably is over about 99% pure. Polypeptide purity or homogeneity isindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a sample, followed by visualizinga single polypeptide band upon staining the gel. For certain purposeshigher resolution can be provided by using HPLC or other means wellknown in the art.

As used herein, the term “non-human animal” refers to any non-humanvertebrate, birds and more usually mammals, preferably primates, farmanimals such as swine, goats, sheep, donkeys, and horses, rabbits orrodents, more preferably rats or mice. As used herein, the term “animal”is used to refer to any vertebrate, preferable a mammal. Both the terms“animal” and “mammal” expressly embrace human subjects unless precededwith the term “non-human”.

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, wherean antibody binding domain is formed from the folding of variabledomains of an antibody molecule to form three-dimensional binding spaceswith an internal surface shape and charge distribution complementary tothe features of an antigenic determinant of an antigen, which allows animmunological reaction with the antigen. Antibodies include recombinantproteins comprising the binding domains, as wells as fragments,including Fab, Fab′, F(ab)₂, and F(ab′)₂ fragments.

As used herein, an “antigenic determinant” is the portion of an antigenmolecule, in this case a CIDE B polypeptide, that determines thespecificity of the antigen-antibody reaction. An “epitope” refers to anantigenic determinant of a polypeptide. An epitope can comprise as fewas 3 amino acids in a spatial conformation which is unique to theepitope. Generally an epitope comprises at least 6 such amino acids, andmore usually at least 8-10 such amino acids. Methods for determining theamino acids which make up an epitope include x-ray crystallography,2-dimensional nuclear magnetic resonance, and epitope mapping e.g. thePepscan method described by Geysen et al. 1984; PCT Publication No. WO84/03564; and PCT Publication No. WO 84/03506.

Throughout the present specification, the expression “nucleotidesequence” may be employed to designate indifferently a polynucleotide ora nucleic acid. More precisely, the expression “nucleotide sequence”encompasses the nucleic material itself and is thus not restricted tothe sequence information (i.e. the succession of letters chosen amongthe four base letters) that biochemically characterizes a specific DNAor RNA molecule.

As used interchangeably herein, the terms “nucleic acids”,“oligonucleotides”, and “polynucleotides” include RNA, DNA, or RNA/DNAhybrid sequences of more than one nucleotide in either single chain orduplex form. The term “nucleotide” as used herein as an adjective todescribe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences ofany length in single-stranded or duplex form. The term “nucleotide” isalso used herein as a noun to refer to individual nucleotides orvarieties of nucleotides, meaning a molecule, or individual unit in alarger nucleic acid molecule, comprising a purine or pyrimidine, aribose or deoxyribose sugar moiety, and a phosphate group, orphosphodiester linkage in the case of nucleotides within anoligonucleotide or polynucleotide. Although the term “nucleotide” isalso used herein to encompass “modified nucleotides” which comprise atleast one modifications (a) an alternative linking group, (b) ananalogous form of purine, (c) an analogous form of pyrimidine, or (d) ananalogous sugar, for examples of analogous linking groups, purine,pyrimidines, and sugars see for example PCT publication No. WO 95/04064.The polynucleotide sequences of the invention may be prepared by anyknown method, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell required to initiate the specific transcription ofa gene.

A sequence which is “operably linked” to a regulatory sequence such as apromoter means that said regulatory element is in the correct locationand orientation in relation to the nucleic acid to control RNApolymerase initiation and expression of the nucleic acid of interest.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. More precisely, twoDNA molecules (such as a polynucleotide containing a promoter region anda polynucleotide encoding a desired polypeptide or polynucleotide) aresaid to be “operably linked” if the nature of the linkage between thetwo polynucleotides does not (1) result in the introduction of aframe-shift mutation or (2) interfere with the ability of thepolynucleotide containing the promoter to direct the transcription ofthe coding polynucleotide.

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by either DNA polymerase, RNApolymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g., polynucleotide as defined hereinbelow) which canbe used to identify a specific polynucleotide sequence present insamples, said nucleic acid segment comprising a nucleotide sequencecomplementary of the specific polynucleotide sequence to be identified.

The terms “trait” and “phenotype” are used interchangeably herein andrefer to any visible, detectable or otherwise measurable property of anorganism such as symptoms of, or susceptibility to a disease forexample. Typically the terms “trait” or “phenotype” are used herein torefer to symptoms of, or susceptibility to a disease, a beneficialresponse to or side effects related to a treatment. Preferably, saidtrait can be, without to be limited to, cancers, developmental diseases,and neurological diseases.

The term “allele” is used herein to refer to variants of a nucleotidesequence. A biallelic polymorphism has two forms. Diploid organisms maybe homozygous or heterozygous for an allelic form.

The term “genotype” as used herein refers the identity of the allelespresent in an individual or a sample. In the context of the presentinvention, a genotype preferably refers to the description of thebiallelic marker alleles present in an individual or a sample. The term“genotyping” a sample or an individual for a biallelic marker involvesdetermining the specific allele or the specific nucleotide carried by anindividual at a biallelic marker.

The term “mutation” as used herein refers to a difference in DNAsequence between or among different genomes or individuals which has afrequency below 1%.

The term “polymorphism” as used herein refers to the occurrence of twoor more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A single nucleotide polymorphism is the replacement ofone nucleotide by another nucleotide at the polymorphic site. Deletionof a single nucleotide or insertion of a single nucleotide also givesrise to single nucleotide polymorphisms. In the context of the presentinvention, “single nucleotide polymorphism” preferably refers to asingle nucleotide substitution. Typically, between differentindividuals, the polymorphic site may be occupied by two differentnucleotides.

The term “biallelic polymorphism” and “biallelic marker” are usedinterchangeably herein to refer to a single nucleotide polymorphismhaving two alleles at a fairly high frequency in the population. A“biallelic marker allele” refers to the nucleotide variants present at abiallelic marker site.

The location of nucleotides in a polynucleotide with respect to thecenter of the polynucleotide are described herein in the followingmanner. When a polynucleotide has an odd number of nucleotides, thenucleotide at an equal distance from the 3′ and 5′ ends of thepolynucleotide is considered to be “at the center” of thepolynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter”, and so on.

Biallelic markers can be defined as genome-derived polynucleotideshaving between 2 and 100, preferably between 20, 30, or 40 and 60, andmore preferably about 47 nucleotides in length, which exhibit biallelicpolymorphism at one single base position. Each biallelic markertherefore corresponds to two forms of a polynucleotide sequence includedin a gene which, when compared with one another, present a nucleotidemodification at one position.

The term “upstream” is used herein to refer to a location which istoward the 5′ end of the polynucleotide from a specific reference point.

The terms “base paired” and “Watson & Crick base paired” are usedinterchangeably herein to refer to nucleotides which can be hydrogenbonded to one another be virtue of their sequence identities in a mannerlike that found in double-helical DNA with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds (See Stryer, 1995).

The terms “complementary” or “complement thereof” are used herein torefer to the sequences of polynucleotides which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. For the purpose ofthe present invention, a first polynucleotide is deemed to becomplementary to a second polynucleotide when each base in the firstpolynucleotide is paired with its complementary base. Complementarybases are, generally, A and T (or A and U), or C and G. “Complement” isused herein as a synonym from “complementary polynucleotide”,“complementary nucleic acid” and “complementary nucleotide sequence”.These terms are applied to pairs of polynucleotides based solely upontheir sequences and not any particular set of conditions under which thetwo polynucleotides would actually bind.

Variants and Fragments

1-Polynucleotides

The invention also relates to variants and fragments of thepolynucleotides described herein.

Variants of polynucleotides, as the term is used herein, arepolynucleotides that differ from a reference polynucleotide. A variantof a polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical.

Nucleotide changes present in a variant polynucleotide may be silent,which means that they do not alter the amino acids encoded by thepolynucleotide.

However, nucleotide changes may also result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions.

In the context of the present invention, particularly preferredembodiments are those in which the polynucleotides encode polypeptideswhich retain substantially the same biological function or activity asthe mature CIDE B protein.

Variants of polynucleotides according to the invention include, withoutbeing limited to, nucleotide sequences at least 95% identical to anucleic acid selected from the group consisting of SEQ ID Nos 1 and 2,and any polynucleotide fragment of at least 8, 20, 50, 75, or 100consecutive nucleotides from a nucleic acid selected from the groupconsisting of SEQ ID Nos 1 and 2, and preferably at least 99% identical,more particularly at least 99.5% identical, and most preferably at least99.8% identical to a nucleic acid selected from the group consisting ofSEQ ID Nos 1 and 2, and any polynucleotide fragment of at least 8, 20,50, 75, or 100 consecutive nucleotides from a nucleic acid selected fromthe group consisting of SEQ ID Nos 1 and 2.

A polynucleotide fragment is a polynucleotide having a sequence that isentirely the same as part but not all of a given nucleotide sequence,preferably the nucleotide sequence of a CIDE B gene, and variantsthereof. The fragment can be a portion of an exon or of an intron of aCIDE B gene. It can also be a portion of the regulatory sequences of theCIDE B gene, preferably of the promoter. Preferably, such fragmentscomprise at least one of the biallelic markers 12-73-49 and 12-74-38 ora biallelic marker in linkage disequilibrium therewith.

Such fragments may be “free-standing”, i.e. not part of or fused toother polynucleotides, or they may be comprised within a single largerpolynucleotide of which they form a part or region. However, severalfragments may be comprised within a single larger polynucleotide.

As representative examples of polynucleotide fragments of the invention,there may be mentioned those which have from about 4, 6, 8, 15, 20, 25,40, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200 nucleotidesin length. Preferred are those fragments having about 47 nucleotides inlength, such as those comprising one of the biallelic markers 12-73-49and 12-74-38. Preferred polynucleotide fragments according to theinvention comprise a contiguous span of at least 35, 40, 50, 60, 70, 80,90, 100, 150, 200, 500, or 1000 nucleotides of one particular nucleicacid.

2-Polypeptides

The invention also relates to variants, fragments, analogs andderivatives of the polypeptides described herein, including mutated CIDEB proteins.

The variant may be 1) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue and such substituted amino acid residue may or may not be oneencoded by the genetic code, or 2) one in which one or more of the aminoacid residues includes a substituent group, or 3) one in which themutated CIDE B is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or 4) one in which the additional amino acids are fused to themutated CIDE B, such as a leader or secretory sequence or a sequencewhich is employed for purification of the mutated CIDE B or a preproteinsequence. Such variants are deemed to be within the scope of thoseskilled in the art.

More particularly, a variant CIDE B polypeptide comprises amino acidchanges ranging from 1, 2, 3, 4, 5, 10 to 20 substitutions, additions ordeletions of one amino acid, preferably from 1 to 10, more preferablyfrom 1 to 5 and most preferably from 1 to 3 substitutions, additions ordeletions of one amino acid. The preferred amino acid changes are thosewhich have little or no influence on the biological activity or thecapacity of the variant CIDE B polypeptide to be recognized byantibodies raised against a native CIDE B protein.

By homologous peptide according to the present invention is meant apolypeptide containing one or several aminoacid additions, deletionsand/or substitutions in the amino acid sequence of a CIDE B polypeptide.In the case of an aminoacid substitution, one or several—consecutive ornon-consecutive—aminoacids are replaced by <<equivalent>> amino acids.

In the case of an amino acid substitution in the amino acid sequence ofa polypeptide according to the invention, one or several amino acids canbe replaced by “equivalent” amino acids. The expression “equivalent”amino acid is used herein to designate any amino acid that may besubstituted for one of the amino acids having similar properties, suchthat one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Generally, the following groups of amino acidsrepresent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn,Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4)Lys, Arg, His; (5) Phe, Tyr, Trp, His.

A specific embodiment of a modified CIDE B peptide molecule of interestaccording to the present invention, includes, but is not limited to, apeptide molecule which is resistant to proteolysis, is a peptide inwhich the —CONH— peptide bond is modified and replaced by a (CH2NH)reduced bond, a (NHCO) retro inverso bond, a (CH2-O) methylene-oxy bond,a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO—CH2)cetomethylene bond, a (CHOH—CH2) hydroxyethylene bond), a (N—N) bound, aE-alcene bond or also a —CH═CH— bond. The invention also encompasses ahuman CIDE B polypeptide or a fragment or a variant thereof in which atleast one peptide bond has been modified as described above.

The polypeptide according to the invention could have post-translationalmodifications. For example, it can present the following modifications:acylation, disulfide bond formation, prenylation, carboxymethylation andphosphorylation.

A polypeptide fragment is a polypeptide having a sequence that entirelyis the same as part but not all of a given polypeptide sequence,preferably a polypeptide encoded by a CIDE B gene and variants thereof.

Such fragments may be “free-standing”, i.e. not part of or fused toother polypeptides, or they may be comprised within a single largerpolypeptide of which they form a part or region. However, severalfragments may be comprised within a single larger polypeptide.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 5, 6, 7, 8, 9 or 10to 15, 10 to 20, 15 to 40, or 30 to 55 amino acids long. Preferredpolypeptide fragments according to the invention comprise a contiguousspan of at least 8 amino acids, preferably at least 10 amino acids, morepreferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 aminoacids of one amino acid sequence. Preferred are those fragmentscontaining at least one amino acid mutation in the CIDE B protein.

Identity between Nucleic Acids or Polypeptides

The terms “percentage of sequence identity” and “percentage homology”are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Homology is evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al.,1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al.,1990; Altschul et al., 1993). In a particularly preferred embodiment,protein and nucleic acid sequence homologies are evaluated using theBasic Local Alignment Search Tool (“BLAST”) which is well known in theart (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993,1997). In particular, five specific BLAST programs are used to performthe following task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.        The BLAST programs identify homologous sequences by identifying        similar segments, which are referred to herein as “high-scoring        segment pairs,” between a query amino or nucleic acid sequence        and a test sequence which is preferably obtained from a protein        or nucleic acid sequence database. High-scoring segment pairs        are preferably identified (i.e., aligned) by means of a scoring        matrix, many of which are known in the art. Preferably, the        scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992;        Henikoff and Henikoff, 1993). Less preferably, the PAM or PAM250        matrices may also be used (see, e.g., Schwartz and Dayhoff,        eds., 1978). The BLAST programs evaluate the statistical        significance of all high-scoring segment pairs identified, and        preferably selects those segments which satisfy a user-specified        threshold of significance, such as a user-specified percent        homology. Preferably, the statistical significance of a        high-scoring segment pair is evaluated using the statistical        significance formula of Karlin (see, e.g., Karlin and Altschul,        1990).

The BLAST programs may be used with the default parameters or withmodified parameters provided by the user.

Stringent Hybridization Conditions

By way of example and not limitation, procedures using conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C., the preferred hybridization temperature,in prehybridization mixture containing 100 μg/ml denatured salmon spermDNA and 5–20×10⁶ cpm of ³²P-labeled probe. Alternatively, thehybridization step can be performed at 65° C. in the presence of SSCbuffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.Subsequently, filter washes can be done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes canbe performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals.Following the wash steps, the hybridized probes are detectable byautoradiography. Other conditions of high stringency which may be usedare well known in the art and cited in Sambrook et al., 1989; andAusubel et al., 1989. These hybridization conditions are suitable for anucleic acid molecule of about 20 nucleotides in length. There is noneed to say that the hybridization conditions described above are to beadapted according to the length of the desired nucleic acid, followingtechniques well known to the one skilled in the art. The suitablehybridizations conditions may for example be adapted according to theteachings disclosed in the book Hames and Higgins (1985) or in Sambrooket al.(1989).

Genomic Sequences of CIDE B

The present invention comprises a purified or isolated nucleic acidencoding the CIDE B polypeptide, wherein said nucleic acid comprisingthe sequence of SEQ ID No 1, a sequence complementary thereto, afragment or a variant thereof.

The invention also encompasses a purified or isolated nucleic acidcomprising a nucleotide sequence having at least 70, 75, 80, 85, 90, or95% nucleotide identity with the nucleotide sequence of SEQ ID No 1, asequence complementary thereto, or a fragment thereof. The nucleotidedifferences as regards to the nucleotide sequences of SEQ ID No 1 aregenerally randomly distributed throughout the entire nucleic acid.Nevertheless, preferred nucleic acids are those wherein the differencesas regards to the nucleotide sequences of SEQ ID No 1 are predominantlylocated outside the coding sequences contained in the exons.

Another object of the invention consists of a purified, isolated, orrecombinant nucleic acid that hybrizes with the sequence of SEQ ID No 1or a complementary sequence thereto or a variant thereof, under thestringent hybridization conditions as defined above.

These nucleic acids, as well as their fragments and variants, may beused as oligonucleotide primers or probes in order to detect thepresence of a copy of the CIDE B gene in a test sample, or alternativelyin order to amplify a target nucleotide sequence within the CIDE Bsequences.

The CIDE B gene has 5 exons. The exon and intron positions in SEQ ID No1 are detailed below in Table A.

TABLE A Position in SEQ ID No 1 Position in SEQ ID No 1 Exon BeginningEnd Intron Beginning End 1 2803 2922 1 2923 3224 2 3225 3369 2 3370 42163 4217 4366 3 4367 4602 4 4603 4793 4 4794 4974 5 4975 5555

Consequently, the invention also concerns a purified or isolated nucleicacid comprising a nucleotide sequence selected from the group consistingof the exons 1, 2, 3, 4, and 5 of the CIDE B gene, a sequencecomplementary thereto, a fragment or a variant thereof.

The invention also deals with a purified or isolated nucleic acidcomprising a combination of at least two polynucleotides selected fromthe group consisting of the exons 1, 2, 3, 4, and 5 of the CIDE B gene,wherein the polynucleotides are ordered within the nucleic acid, fromthe 5′ end to the 3′ end of said nucleic acid, in the same order than inthe SEQ ID No 1.

Thus, the invention embodies purified, isolated, or recombinantpolynucleotides comprising a nucleotide sequence selected from the groupconsisting of the introns of the CIDE B gene, or a sequencecomplementary thereto.

Particularly preferred nucleic acids of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides of the nucleotide sequence of SEQ ID No 1, or thecomplements thereof. Additionally preferred nucleic acids of theinvention include isolated, purified, or recombinant polynucleotidescomprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40,50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No1 or the complements thereof, wherein said contiguous span comprises atleast 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ IDNo 1: 1–1000, 1001–2000, 2001–3000, 3001–4000, 4001–5000, 5001–6000,6001–7000, 7001–8000, 8001–9000, 9001–10000, 10001–10961.

While this section is entitled “Genomic Sequences of CIDE B,” it shouldbe noted that nucleic acid fragments of any size and sequence may alsobe comprised by the polynucleotides described in this section, flankingthe genomic sequences of CIDE B on either side or between two or moresuch genomic sequences.

CIDE B cDNA Sequences

The inventors have discovered that the expression of the CIDE B geneleads to the production of at least one mRNA molecule, the nucleic acidsequence of which is set forth in SEQ ID No 2.

Another object of the invention consists of a purified or isolatednucleic acid comprising the nucleotide sequence of SEQ ID No 2 orfragments or variants thereof, or a complementary sequence thereto.

The invention also pertains to a purified or isolated nucleic acidhaving at least 70, 75, 80, 85, 90, or 95% nucleotide identity with thenucleotide sequence of SEQ ID No 2, a sequence complementary thereto, ora fragment thereof.

Another object of the invention consists of a purified, isolated, orrecombinant nucleic acid that hybridizes with the sequence of SEQ ID No2 or a complementary sequence thereto or a variant thereof, under thestringent hybridization conditions as defined above.

The nucleotide differences as regards to the nucleotide sequence of SEQID No 2 are generally randomly distributed throughout the entire nucleicacid. Nevertheless, preferred nucleic acids are those wherein thenucleotide differences as regards to the nucleotide sequence of SEQ IDNo 2 are predominantly located outside the coding sequences, and moreprecisely in the 5′-UTR and the 3′-UTR sequences contained in thenucleotide sequence of SEQ ID No 2.

The cDNA of SEQ ID No 2 includes a 5′-UTR region starting from thenucleotide at position 1 and ending at the nucleotide in position 79 ofSEQ ID No 2. The cDNA of SEQ ID No 2 includes a 3′-UTR region startingfrom the nucleotide at position 740 and ending at the nucleotide atposition 1187 of SEQ ID No 2.

Consequently, the invention concerns a purified or isolated nucleic acidcomprising a nucleotide sequence selected from a group consisting of the5′UTR and 3′UTR of the CIDE B cDNA, a sequence complementary thereto, afragment or a variant thereof.

The middle line of FIG. 1 depicts the main structural features of apurified or isolated nucleic acid consisting of a CIDE B cDNA. The5′-end sequence of this cDNA, more particularly the nucleotide sequencecomprised between the nucleotide in position 1 and the nucleotide inposition 247 of the nucleic acid of SEQ ID No 2 molecule corresponds tothe nucleotide sequence of a 5′-EST that has been obtained from a humanliver cDNA library. This 5′-EST is also part of the invention.

The invention also relates to isolated, purified, or recombinantpolynucleotides comprising a contiguous span of at least 35, 40, 50, 60,70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of the nucleotidesequence of SEQ ID No 2, or the complements thereof. Particularlypreferred nucleic acids of the invention include isolated, purified, orrecombinant polynucleotides comprising a contiguous span of at least 12,15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein saidcontiguous span comprises at least 1, 2, 3, 5, or 10 of the followingnucleotide positions of SEQ ID No 2: 1–78, 91–190, 208–229, 243–288,301–328, 364–394, 409–457, 478–490, 505–508, 529–597, 616–633,656–667,682–688,703–1188.

While this section is entitled “CIDE B cDNA Sequences,” it should benoted that nucleic acid fragments of any size and sequence may also becomprised by the polynucleotides described in this section, flanking thegenomic sequences of CIDE B on either side or between two or more suchgenomic sequences.

Coding Regions of CIDE B

The CIDE B open reading frame is contained in the corresponding mRNA ofSEQ ID No 2 and is a further object of the present invention.

More precisely, the effective CIDE B coding sequence (CDS) is comprisedbetween the nucleotide at position 80 (first nucleotide of the ATGcodon) and the nucleotide at position 739 (end nucleotide of the TAAcodon) of SEQ ID No 2. A purified or isolated polynucleotide comprisingthe CIDE B coding region defined above is another object of theinvention.

The present invention concerns a purified or isolated nucleic acidencoding a human CIDE B protein, wherein said CIDE B protein comprisesan amino acid sequence of SEQ ID No 3, a nucleotide sequencecomplementary thereto, a fragment or a variant thereof. The presentinvention also embodies isolated, purified, and recombinantpolynucleotides which encode a polypeptides comprising a contiguous spanof at least 35, 40, 50, or 100 amino acids of SEQ ID No 3. In apreferred embodiment, the present invention embodies isolated, purified,and recombinant polynucleotides which encode a polypeptides comprising acontiguous span of at least 8 amino acids, preferably at least 10 aminoacids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or200 amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 1–29,47–70, 103–115, 124, 134, 169–185, and 203–219. In an additionalpreferred embodiment, the present invention embodies isolated, purified,and recombinant polynucleotides which encode a polypeptides comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100,150 or 200 amino acids of SEQ ID No 3, wherein said contiguous spanincludes at least 1, 2, 3, 5 or of the following amino acid positions:7–11, 18–29, 47, 55–63, 70, 103–104, 111–115, 124, 134, 169–173,181–185, and 203–219.

The above disclosed polynucleotide that contains the coding sequence ofthe CIDE B gene of the invention may be expressed in a desired host cellor a desired host organism, when this polynucleotide is placed under thecontrol of suitable expression signals. The expression signals may beeither the expression signals contained in the regulatory regions in theCIDE B gene of the invention or in contrast be exogenous regulatorynucleic sequences. Such a polynucleotide, when placed under the suitableexpression signals, may also be inserted in a vector for its expression.

CIDE B Regulatory Sequences

As already mentioned hereinbefore, the genomic sequence of the CIDE Bgene contains regulatory sequences both in the non-coding 5′-flankingregion and in the non-coding 3′-flanking region that border the CIDE Bcoding region containing the exons of this gene.

The 5′-regulatory sequence of the CIDE B gene comprises the nucleotidesequence which is localized between the nucleotide in position 1 and thenucleotide in position 2802 of the nucleotide sequence of SEQ ID No 1.This polynucleotide would contain the promoter site.

The 3′-regulatory sequence of the CIDE B gene comprises the nucleotidesequence which is localized between the nucleotide in position 5556 andthe nucleotide in position 10961 of the nucleotide sequence of SEQ ID No1.

Polynucleotides derived from the CIDE B regulatory regions describedabove are useful in order to detect the presence of at least a copy of anucleotide sequence of SEQ ID No 1, or a fragment or a variant thereofin a test sample.

Thus, the present invention also concerns a purified or isolated nucleicacid comprising a polynucleotide which is selected from the groupconsisting of the 5′ and 3′ regulatory regions, or a sequencecomplementary thereto or a biologically active fragment or variantthereof. “5′ regulatory region” refers to the nucleotide sequencelocated between positions 1 and 2802 of SEQ ID No 1. “3′ regulatoryregion” refers to the nucleotide sequence located between positions 5556and 10961 of SEQ ID No 1.

The invention also pertains to a purified or isolated nucleic acidcomprising a polynucleotide having at least 70, 75, 80, 85, 90, or 95%nucleotide identity with the nucleotide sequence selected from the groupconsisting of the 5′ and 3′ regulatory regions, or a sequencecomplementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated orrecombinant nucleic acids comprising a polynucleotide that hybridizes,under the stringent hybridization conditions defined herein, with apolynucleotide selected from the group consisting of the nucleotidesequences of the 5′- and 3′ regulatory regions, or a sequencecomplementary thereto or a variant thereof or a biologically activefragment thereof.

The promoter activity of the regulatory regions contained in the 5′regulatory region of CIDE B can be assessed as described below.

In order to identify the relevant biologically active polynucleotidefragments or variants of the 5′ and 3′ regulatory regions, the one skillin the art will refer to the book of Sambrook et al. (1989) whichdescribes the use of a recombinant vector carrying a marker gene (i.e.beta galactosidase, chloramphenicol acetyl transferase, etc.) theexpression of which will be detected when placed under the control of abiologically active polynucleotide fragments or variants of the 5′ and3′ regulatory regions. Genomic sequences located upstream of the firstexon of the CIDE B gene are cloned into a suitable promoter reportervector, such as the pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic,pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available fromClontech, or pGL2-basic or pGL3-basic promoterless luciferase reportergene vector from Promega. Briefly, each of these promoter reportervectors include multiple cloning sites positioned upstream of a reportergene encoding a readily assayable protein such as secreted alkalinephosphatase, luciferase, beta galactosidase, or green fluorescentprotein. The sequences upstream the CIDE B coding region are insertedinto the cloning sites upstream of the reporter gene in bothorientations and introduced into an appropriate host cell. The level ofreporter protein is assayed and compared to the level obtained from avector which lacks an insert in the cloning site. The presence of anelevated expression level in the vector containing the insert withrespect to the control vector indicates the presence of a promoter inthe insert. If necessary, the upstream sequences can be cloned intovectors which contain an enhancer for increasing transcription levelsfrom weak promoter sequences. A significant level of expression abovethat observed with the vector lacking an insert indicates that apromoter sequence is present in the inserted upstream sequence.

Promoter sequences within the upstream genomic DNA may be furtherdefined by constructing nested 5′ and/or 3′ deletions in the upstreamDNA using conventional techniques such as Exonuclease III or appropriaterestriction endonuclease digestion. The resulting deletion fragments canbe inserted into the promoter reporter vector to determine whether thedeletion has reduced or obliterated promoter activity, such asdescribed, for example, by Coles et al. (1998). In this way, theboundaries of the promoters may be defined. If desired, potentialindividual regulatory sites within the promoter may be identified usingsite directed mutagenesis or linker scanning to obliterate potentialtranscription factor binding sites within the promoter individually orin combination. The effects of these mutations on transcription levelsmay be determined by inserting the mutations into cloning sites inpromoter reporter vectors. This type of assay is well-known to thoseskilled in the art and is described in WO 97/17359, U.S. Pat. No.5,374,544, EP 582 796, U.S. Pat. Nos. 5,698,389, 5,643,746, 5,502,176,and U.S. Pat. No. 5,266,488.

The strength and the specificity of the promoter of the CIDE B gene canbe assessed through the expression levels of a detectable polynucleotideoperably linked to the CIDE B promoter in different types of cells andtissues. The detectable polynucleotide may be either a polynucleotidethat specifically hybridizes with a predefined oligonucleotide probe, ora polynucleotide encoding a detectable protein, including a CIDE Bpolypeptide or a fragment or a variant thereof. This type of assay iswell-known to those skilled in the art and is described in U.S. Pat.Nos. 5,502,176, and 5,266,488.

Polynucleotides carrying the 5′ and 3′ regulatory regions of CIDE Bcoding region may be advantageously used to control the transcriptionaland translational activity of an heterologous polynucleotide ofinterest.

Thus, the present invention also concerns a purified or isolated nucleicacid comprising a polynucleotide which is selected from the groupconsisting of the 5′ and 3′ regulatory regions of CIDE B, or a sequencecomplementary thereto or a biologically active fragment or variantthereof.

Preferred fragments of the 5′ regulatory region of CIDE B have a lengthof about 1000 nucleotides, more particularly about 500 nucleotides, morepreferably 200 nucleotides and most preferably about 100 nucleotides.

Preferred fragments of 3′ regulatory region of CIDE B have a length ofabout 1000 nucleotides, more particularly about 500 nucleotides, morepreferably 200 nucleotides and most preferably about 100 nucleotides.

By a “biologically active” polynucleotide derivative of the 5′ and 3′regulatory regions of the CIDE B is intended a polynucleotide comprisingor alternatively consisting in a fragment of said polynucleotide whichis functional as a regulatory region for expressing a recombinantpolypeptide or a recombinant polynucleotide in a recombinant cell host.

For the purpose of the invention, a nucleic acid or polynucleotide is“functional” as a regulatory region for expressing a recombinantpolypeptide or a recombinant polynucleotide if said regulatorypolynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operably linked” to nucleotide sequences which encode thedesired polypeptide or the desired polynucleotide.

These regulatory polynucleotides can also be prepared by nucleic acidchemical synthesis, as described elsewhere in the specification, whereoligonucleotide probes or primers synthesis is disclosed.

The regulatory polynucleotides according to the invention may beadvantageously part of a recombinant expression vector that may be usedto express a coding sequence in a desired host cell or host organism.The recombinant expression vectors according to the invention aredescribed elsewhere in the specification.

A preferred 5′-regulatory polynucleotide of the invention includes the5′-UTR of CIDE B, or a biologically active fragment or variant thereof.

A preferred 3′-regulatory polynucleotide of the invention includes a3′-UTR of CIDE B, or a biologically active fragment or variant thereof.This preferred 3′-regulatory polynucleotide carries a polyadenylationsite located between the nucleotide in position 1158 and the nucleotidein position 1163 of the nucleic acid of SEQ ID No 2.

The regulatory polynucleotides of the invention may be prepared from anyof the nucleotide sequence of SEQ ID No 1 by cleavage using suitablerestriction enzymes, as described for example in the book of Sambrook etal. (1989). The regulatory polynucleotides may also be prepared bydigestion of any of SEQ ID No 1 by an exonuclease enzyme, such as forexample Bal31 (Wabiko et al., 1986).

A further object of the invention consists of a purified or isolatednucleic acid comprising:

-   -   a) a nucleic acid comprising the 5′ regulatory region of CIDE B        or a biologically active fragment or variant thereof;    -   b) a polynucleotide encoding a desired polypeptide or nucleic        acid operably linked to said 5′ regulatory polynucleotide or its        biologically active fragment or variant thereof;    -   c) optionally, a nucleic acid comprising the 3′ regulatory        region of CIDE B or a biologically active fragment or variant        thereof.

In a specific embodiment of the nucleic acid defined above, said nucleicacid includes the 5′-UTR of CIDE B, or a biologically active fragment orvariant thereof. In a second specific embodiment of the nucleic aciddefined above, said nucleic acid includes the 3′-UTR of CIDE B, or abiologically active fragment or variant thereof.

The 5′ regulatory region of CIDE B, or its biologically active fragmentsor variants, is advantageously operably linked at the 5′-end of thepolynucleotide encoding the desired polypeptide or polynucleotide.

The 3′ regulatory region of CIDE B, or its biologically active fragmentsand variants, is advantageously placed at the 3′-end of thepolynucleotide encoding the desired polypeptide or polynucleotide.

The desired polypeptide encoded by the above described nucleic acid maybe of various nature or origin, encompassing proteins of prokaryotic oreukaryotic origin. Among the polypeptides expressed under the control ofa CIDE B regulatory region, there may be cited bacterial, fungal orviral antigens. Also encompassed are eukaryotic proteins such asintracellular proteins, like “house keeping” proteins, membrane-boundproteins, like receptors, and secreted proteins like the numerousendogenous mediators such as cytokines. The desired polypeptide may bethe CIDE B protein, especially the protein of the amino acid sequence ofSEQ ID No 3, or a fragment or a variant thereof.

The desired nucleic acids encoded by the above described polynucleotide,usually a RNA molecule, may be complementary to a desired codingpolynucleotide, for example to the CIDE B coding sequence, and thususeful as an antisense polynucleotide.

Such a polynucleotide may be included in a recombinant expression vectorin order to express the desired polypeptide or the desired nucleic acidin host cell or in a host organism. Suitable recombinant vectors thatcontain a polynucleotide such as described hereinbefore are disclosedelsewhere in the specification.

Oligonucleotide Probes and Primers

Polynucleotides derived from the CIDE B gene are useful in order todetect the presence of at least a copy of a nucleotide sequence of SEQID No 1 or 2, or a fragment, complement, or variant thereof in a testsample.

Particularly preferred probes and primers of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof.Further preferred probes and primers of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof,wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of thefollowing nucleotide positions of SEQ ID No 1: 1–1000, 1001–2000,2001–3000, 3001–4000, 4001–5000, 5001–6000, 6001–7000, 7001–8000,8001–9000, 9001–10000, 10001–10961.

Other preferred probes and primers of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides of SEQ ID No 2 or the complements thereof. Additionalpreferred probes and primers of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof,wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of thefollowing nucleotide positions of SEQ ID No 2: 1–78, 91–190, 208–229,243–288, 301–328, 364–394, 409–457, 478–490, 505–508, 529–597, 616–633,656–667, 682–688, 703–1188.

Thus, the invention also relates to nucleic acid probes characterized inthat they hybridize specifically, under the stringent hybridizationconditions defined above, with a nucleic acid selected from the groupconsisting of the nucleotide sequences of SEQ ID Nos 1 and 2 or avariant thereof or a sequence complementary thereto.

In one embodiment the invention encompasses isolated, purified, andrecombinant polynucleotides consisting of, or consisting essentially ofa contiguous span of 8 to 50 nucleotides of any one of SEQ ID No 1 andthe complement thereof, wherein said span includes a CIDE B -relatedbiallelic marker in said sequence; optionally, wherein said CIDE B-related biallelic marker is selected from the group consisting of thebiallelic markers 12-73-49 and 12-74-38, and the complements thereof;optionally, wherein said contiguous span is 18 to 47 nucleotides inlength and said biallelic marker is within 4 nucleotides of the centerof said polynucleotide; optionally, wherein said polynucleotide consistsof said contiguous span and said contiguous span is 25 nucleotides inlength and said biallelic marker is at the center of saidpolynucleotide; optionally, wherein the 3′ end of said contiguous spanis present at the 3′ end of said polynucleotide; and optionally, whereinthe 3′ end of said contiguous span is located at the 3′ end of saidpolynucleotide and said biallelic marker is present at the 3′ end ofsaid polynucleotide. In a preferred embodiment, said probes comprises,consists of, or consists essentially of a sequence selected from thefollowing sequences: P(12-73-49) and P(12-74-38) and the complementarysequences thereto.

In another embodiment the invention encompasses isolated, purified andrecombinant polynucleotides comprising, consisting of, or consistingessentially of a contiguous span of 8 to 50 nucleotides of SEQ ID No 1,or the complements thereof, wherein the 3′ end of said contiguous spanis located at the 3′ end of said polynucleotide, and wherein the 3′ endof said polynucleotide is located within 20 nucleotides upstream of aCIDE B -related biallelic marker in said sequence; optionally, whereinsaid CIDE B -related biallelic marker is selected from the groupconsisting of the biallelic markers 12-73-49 and 12-74-38, and thecomplements thereof; optionally, wherein the 3′ end of saidpolynucleotide is located 1 nucleotide upstream of said CIDE B -relatedbiallelic marker in said sequence; and optionally, wherein saidpolynucleotide consists essentially of a sequence selected from thefollowing sequences: D(12-73-49), D(12-74-38), E(12-73-49), andE(12-74-38).

In a further embodiment, the invention encompasses isolated, purified,or recombinant polynucleotides comprising, consisting of, or consistingessentially of a sequence selected from the following sequences:B(12-73), B(12-74), C(12-73), and C(12-74).

In an additional embodiment, the invention encompasses polynucleotidesfor use in hybridization assays, sequencing assays, and enzyme-basedmismatch detection assays for determining the identity of the nucleotideat a CIDE B -related biallelic marker in SEQ ID No 1 or the complementsthereof, as well as polynucleotides for use in amplifying segments ofnucleotides comprising a CIDE B -related biallelic marker in SEQ ID No 1or the complements thereof; optionally, wherein said CIDE B -relatedbiallelic marker is selected from the group consisting of the biallelicmarkers 12-73-49 and 12-74-38, and the complements thereof.

A probe or a primer according to the invention has between 8 and 1000nucleotides in length, or is specified to be at least 12, 15, 18, 20,25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length.More particularly, the length of these probes and primers can range from8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, morepreferably from 15 to 30 nucleotides. Shorter probes and primers tend tolack specificity for a target nucleic acid sequence and generallyrequire cooler temperatures to form sufficiently stable hybrid complexeswith the template. Longer probes and primers are expensive to produceand can sometimes self-hybridize to form hairpin structures. Theappropriate length for primers and probes under a particular set ofassay conditions may be empirically determined by one of skill in theart. A preferred probe or primer consists of a nucleic acid comprising apolynucleotide selected from the group of the nucleotide sequences ofP(12-73-49) and P(12-74-38) and the complementary sequence thereto,B(12-73), B(12-74), C(12-73), C(12-74), D(12-73-49), D(12-74-38), forwhich the respective locations in the sequence listing are provided inTables 1, 2, 3 and 4.

The formation of stable hybrids depends on the melting temperature (Tm)of the DNA. The Tm depends on the length of the primer or probe, theionic strength of the solution and the G+C content. The higher the G+Ccontent of the primer or probe, the higher is the melting temperaturebecause G:C pairs are held by three H bonds whereas A:T pairs have onlytwo. The GC content in the probes of the invention usually rangesbetween 10 and 75%, preferably between 35 and 60%, and more preferablybetween 40 and 55%.

The primers and probes can be prepared by any suitable method,including, for example, cloning and restriction of appropriate sequencesand direct chemical synthesis by a method such as the phosphodiestermethod of Narang et al.(1979), the phosphodiester method of Brown etal.(1979), the diethylphosphoramidite method of Beaucage et al.(1981)and the solid support method described in EP 0 707 592.

Detection probes are generally nucleic acid sequences or unchargednucleic acid analogs such as, for example peptide nucleic acids whichare disclosed in International Patent application WO 92/20702,morpholino analogs which are described in U.S. Pat. Nos. 5,185,444;5,034,506 and 5,142,047. The probe may have to be rendered“non-extendable” in that additional dNTPs cannot be added to the probe.In and of themselves analogs usually are non-extendable and nucleic acidprobes can be rendered non-extendable by modifying the 3′ end of theprobe such that the hydroxyl group is no longer capable of participatingin elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified, U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 describes modifications, whichcan be used to render a probe non-extendable.

Any of the polynucleotides of the present invention can be labeled, ifdesired, by incorporating any label known in the art to be detectable byspectroscopic, photochemical, biochemical, immumunochemical, or chemicalmeans. For example, useful labels include radioactive substances(including, ³²p, ³⁵S, ³H, ¹²⁵I), fluorescent dyes (including,5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) orbiotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends.Examples of non-radioactive labeling of nucleic acid fragments aredescribed in the French patent No. FR-7810975 or by Urdea et al (1988)or Sanchez-Pescador et al (1988). In addition, the probes according tothe present invention may have structural characteristics such that theyallow the signal amplification, such structural characteristics being,for example, branched DNA probes as those described by Urdea et al. in1991 or in the European patent No. EP 0 225 807 (Chiron).

A label can also be used to capture the primer, so as to facilitate theimmobilization of either the primer or a primer extension product, suchas amplified DNA, on a solid support. A capture label is attached to theprimers or probes and can be a specific binding member which forms abinding pair with the solid's phase reagent's specific binding member(e.g. biotin and streptavidin). Therefore depending upon the type oflabel carried by a polynucleotide or a probe, it may be employed tocapture or to detect the target DNA. Further, it will be understood thatthe polynucleotides, primers or probes provided herein, may, themselves,serve as the capture label. For example, in the case where a solid phasereagent's binding member is a nucleic acid sequence, it may be selectedsuch that it binds a complementary portion of a primer or probe tothereby immobilize the primer or probe to the solid phase. In caseswhere a polynucleotide probe itself serves as the binding member, thoseskilled in the art will recognize that the probe will contain a sequenceor “tail” that is not complementary to the target. In the case where apolynucleotide primer itself serves as the capture label, at least aportion of the primer will be free to hybridize with a nucleic acid on asolid phase. DNA Labeling techniques are well known to the skilledtechnician.

The probes of the present invention are useful for a number of purposes.They can be notably used in Southern hybridization to genomic DNA. Theprobes can also be used to detect PCR amplification products. They mayalso be used to detect mismatches in the CIDE B gene or mRNA using othertechniques.

Any of the polynucleotides, primers and probes of the present inventioncan be conveniently immobilized on a solid support. Solid supports areknown to those skilled in the art and include the walls of wells of areaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, duracytes andothers. The solid support is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and duracytes are all suitable examples. Suitable methods forimmobilizing nucleic acids on solid phases include ionic, hydrophobic,covalent interactions and the like. A solid support, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid support can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid support and which has the ability to immobilize thecapture reagent through a specific binding reaction. The receptormolecule enables the indirect binding of the capture reagent to a solidsupport material before the performance of the assay or during theperformance of the assay. The solid phase thus can be a plastic,derivatized plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microliter well, sheet, bead, microparticle,chip, sheep (or other suitable animal's) red blood cells, duracytes® andother configurations known to those of ordinary skill in the art. Thepolynucleotides of the invention can be attached to or immobilized on asolid support individually or in groups of at least 2, 5, 8, 10, 12, 15,20, or 25 distinct polynucleotides of the invention to a single solidsupport. In addition, polynucleotides other than those of the inventionmay be attached to the same solid support as one or more polynucleotidesof the invention.

Consequently, the invention also comprises a method for detecting thepresence of a nucleic acid comprising a nucleotide sequence selectedfrom a group consisting of SEQ ID Nos 1 and 2, a fragment or a variantthereof and a complementary sequence thereto in a sample, said methodcomprising the following steps of:

-   -   a) bringing into contact a nucleic acid probe or a plurality of        nucleic acid probes which can hybridize with a nucleotide        sequence included in a nucleic acid selected form the group        consisting of the nucleotide sequences of SEQ ID Nos 1 and 2, a        fragment or a variant thereof and a complementary sequence        thereto and the sample to be assayed; and    -   b) detecting the hybrid complex formed between the probe and a        nucleic acid in the sample.

The invention further concerns a kit for detecting the presence of anucleic acid comprising a nucleotide sequence selected from a groupconsisting of SEQ ID Nos 1 and 2, a fragment or a variant thereof and acomplementary sequence thereto in a sample, said kit comprising:

-   -   a) a nucleic acid probe or a plurality of nucleic acid probes        which can hybridize with a nucleotide sequence included in a        nucleic acid selected form the group consisting of the        nucleotide sequences of SEQ ID Nos 1 and 2, a fragment or a        variant thereof and a complementary sequence thereto; and    -   b) optionally, the reagents necessary for performing the        hybridization reaction.

In a first preferred embodiment of this detection method and kit, saidnucleic acid probe or the plurality of nucleic acid probes are labeledwith a detectable molecule. In a second preferred embodiment of saidmethod and kit, said nucleic acid probe or the plurality of nucleic acidprobes has been immobilized on a substrate. In a third preferredembodiment, the nucleic acid probe or the plurality of nucleic acidprobes comprise either a sequence which is selected from the groupconsisting of the nucleotide sequences of P(12-73-49) and P(12-74-38)and the complementary sequence thereto, B(12-73), B(12-74), C(12-73),C(12-74), D(12-73-49), D(12-74-38), E(12-73-49), and E(12-74-38) or abiallelic marker selected from the group consisting of the biallelicmarkers 12-73–49 and 12-74-38 and the complements thereto.

Oligonucleotide Arrays

A substrate comprising a plurality of oligonucleotide primers or probesof the invention may be used either for detecting or amplifying targetedsequences in the CIDE B gene and may also be used for detectingmutations in the coding or in the non-coding sequences of the CIDE Bgene.

Any polynucleotide provided herein may be attached in overlapping areasor at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention. One particular embodiment of thesepolynucleotide arrays is known as the Genechips™, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092. These arrays may generally be produced using mechanicalsynthesis methods or light directed synthesis methods which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis (Fodor et al., 1991). The immobilization ofarrays of oligonucleotides on solid supports has been rendered possibleby the development of a technology generally identified as “Very LargeScale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically,probes are immobilized in a high density array on a solid surface of achip. Examples of VLSIPS™ technologies are provided in U.S. Pat. Nos.5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO92/10092 and WO 95/11995, which describe methods for formingoligonucleotide arrays through techniques such as light-directedsynthesis techniques. In designing strategies aimed at providing arraysof nucleotides immobilized on solid supports, further presentationstrategies were developed to order and display the oligonucleotidearrays on the chips in an attempt to maximize hybridization patterns andsequence information. Examples of such presentation strategies aredisclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 andWO 97/31256.

In another embodiment of the oligonucleotide arrays of the invention, anoligonucleotide probe matrix may advantageously be used to detectmutations occurring in the CIDE B gene and preferably in its regulatoryregion. For this particular purpose, probes are specifically designed tohave a nucleotide sequence allowing their hybridization to the genesthat carry known mutations (either by deletion, insertion orsubstitution of one or several nucleotides). By known mutations, it ismeant, mutations on the CIDE B gene that have been identified according,for example to the technique used by Huang et al.(1996) or Samson etal.(1996).

Another technique that is used to detect mutations in the CIDE B gene isthe use of a high-density DNA array. Each oligonucleotide probeconstituting a unit element of the high density DNA array is designed tomatch a specific subsequence of the CIDE B genomic DNA or cDNA. Thus, anarray consisting of oligonucleotides complementary to subsequences ofthe target gene sequence is used to determine the identity of the targetsequence with the wild gene sequence, measure its amount, and detectdifferences between the target sequence and the reference wild genesequence of the CIDE B gene. In one such design, termed 4L tiled array,is implemented a set of four probes (A, C, G, T), preferably15-nucleotide oligomers. In each set of four probes, the perfectcomplement will hybridize more strongly than mismatched probes.Consequently, a nucleic acid target of length L is scanned for mutationswith a tiled array containing 4L probes, the whole probe set containingall the possible mutations in the known wild reference sequence. Thehybridization signals of the 15-mer probe set tiled array are perturbedby a single base change in the target sequence. As a consequence, thereis a characteristic loss of signal or a “footprint” for the probesflanking a mutation position. This technique was described by Chee etal. in 1996.

Consequently, the invention concerns an array of nucleic acid moleculescomprising at least one polynucleotide described above as probes andprimers. Preferably, the invention concerns an array of nucleic acidcomprising at least two polynucleotides described above as probes andprimers.

A further object of the invention consists of an array of nucleic acidsequences comprising either at least one of the sequences selected fromthe group consisting of P(12-73-49), P(12-74-38), B(12-73), B(12-74),C(12-73), C(12-74), D(12-73-49), and E(12-74-38), the sequencescomplementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18,20, 25, 30, or 40 consecutive nucleotides thereof, and at least onesequence comprising a biallelic marker selected from the groupconsisting of the biallelic markers 12-73-49 and 12-74–38 and thecomplements thereto.

The invention also pertains to an array of nucleic acid sequencescomprising either at least two of the sequences selected from the groupconsisting of P(12-73-49), P(12-74–38), B(12-73), B(12-74), C(12-73),C(12-74), D(12-73-49), D(12-74-38), E(12-73-49), P(12-74-38), thesequences complementary thereto, a fragment thereof of at least 8consecutive nucleotides thereof, and at least two sequences comprising abiallelic marker selected from the group consisting of the biallelicmarkers 12-73-49 and 12-74-38 and the complements thereof.

Amplification of the CIDE B Gene

DNA Extraction

As for the source of the genomic DNA to be subjected to analysis, anytest sample can be foreseen without any particular limitation. Thesetest samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants; fixed tissue specimens including tumor andnon-tumor tissue and lymph node tissues; bone marrow aspirates and fixedcell specimens. The preferred source of genomic DNA used in the contextof the present invention is from peripheral venous blood of each donor.

The techniques of DNA extraction are well-known to the skilledtechnician. Such techniques are described notably by Lin et al. (1998)and by Mackey et al. (1998).

DNA Amplification

Amplification techniques that can be used in the context of the presentinvention include, but are not limited to, the ligase chain reaction(LCR) described in EP-A- 320 308, WO 9320227 and EP-A-439 182, thepolymerase chain reaction (PCR, RT-PCR) and techniques such as thenucleic acid sequence based amplification (NASBA) described in GuatelliJ. C., et al.(1990) and in Compton J.(1991), Q-beta amplification asdescribed in European Patent Application No 4544610, strand displacementamplification as described in Walker et al.(1996) and EP A 684 315 and,target mediated amplification as described in PCT Publication WO9322461.

LCR and Gap LCR are exponential amplification techniques, both depend onDNA ligase to join adjacent primers annealed to a DNA molecule. InLigase Chain Reaction (LCR), probe pairs are used which include twoprimary (first and second) and two secondary (third and fourth) probes,all of which are employed in molar excess to target. The first probehybridizes to a first segment of the target strand and the second probehybridizes to a second segment of the target strand, the first andsecond segments being contiguous so that the primary probes abut oneanother in 5′ phosphate-3′hydroxyl relationship, and so that a ligasecan covalently fuse or ligate the two probes into a fused product. Inaddition, a third (secondary) probe can hybridize to a portion of thefirst probe and a fourth (secondary) probe can hybridize to a portion ofthe second probe in a similar abutting fashion. Of course, if the targetis initially double stranded, the secondary probes also will hybridizeto the target complement in the first instance. Once the ligated strandof primary probes is separated from the target strand, it will hybridizewith the third and fourth probes, which can be ligated to form acomplementary, secondary ligated product. It is important to realizethat the ligated products are functionally equivalent to either thetarget or its complement. By repeated cycles of hybridization andligation, amplification of the target sequence is achieved. A method formultiplex LCR has also been described (WO 9320227). Gap LCR (GLCR) is aversion of LCR where the probes are not adjacent but are separated by 2to 3 bases.

For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR(RT-AGLCR) as described by Marshall et al.(1994). AGLCR is amodification of GLCR that allows the amplification of RNA.

The PCR technology is the preferred amplification technique used in thepresent invention. A variety of PCR techniques are familiar to thoseskilled in the art. For a review of PCR technology, see White (1997) andthe publication entitled “PCR Methods and Applications” (1991, ColdSpring Harbor Laboratory Press). In each of these PCR procedures, PCRprimers on either side of the nucleic acid sequences to be amplified areadded to a suitably prepared nucleic acid sample along with dNTPs and athermostable polymerase such as Taq polymerase, Pfu polymerase, or Ventpolymerase. The nucleic acid in the sample is denatured and the PCRprimers are specifically hybridized to complementary nucleic acidsequences in the sample. The hybridized primers are extended.Thereafter, another cycle of denaturation, hybridization, and extensionis initiated. The cycles are repeated multiple times to produce anamplified fragment containing the nucleic acid sequence between theprimer sites. PCR has further been described in several patentsincluding U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188.

The present invention also relates to a method for the amplification ofa human CIDE B gene sequence, particularly of a portion of the genomicsequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID No 2, or avariant thereof in a test sample, said method comprising the steps of:

-   -   a) contacting a test sample suspected of containing the targeted        CIDE B gene sequence comprised in a nucleotide sequence selected        from a group consisting of SEQ ID Nos 1 and 2, or fragments or        variants thereof with amplification reaction reagents comprising        a pair of amplification primers as described above and located        on either side of the polynucleotide region to be amplified, and    -   b) optionally, detecting the amplification products.

The invention also concerns a kit for the amplification of a human CIDEB gene sequence, particularly of a portion of the genomic sequence ofSEQ ID No 1 or of the cDNA sequence of SEQ ID No 2, or a variant thereofin a test sample, wherein said kit comprises:

-   -   a) a pair of oligonucleotide primers located on either side of        the CIDE B region to be amplified;    -   b) Optionally, the reagents necessary for performing the        amplification reaction.

In one specific embodiment of the above amplification method and kit,the -74), C(12-73), C(12-74), D(12-73-49), D(12-74-38), E(12-73–49), and#(12-74-38).

In another embodiment of the above amplification method and kit, theamplification product is detected by hybridization with a labeled probehaving a sequence which is complementary to the amplified region.

CIDE B Polypeptide and Peptide Fragments Thereof

It is now easy to produce proteins in high amounts by geneticengineering techniques through expression vectors such as plasmids,phages or phagemids. The polynucleotide that code for one thepolypeptides of the present invention is inserted in an appropriateexpression vector in order to produce the polypeptide of interest invitro.

Thus, the present invention also concerns a method for producing one ofthe polypeptides described herein, and especially a polypeptide of SEQID No 3 or a fragment or a variant thereof, wherein said methodcomprises the steps of:

-   -   a) culturing, in an appropriate culture medium, a cell host        previously transformed or transfected with the recombinant        vector comprising a nucleic acid encoding a CIDE B polypeptide,        or a fragment or a variant thereof;    -   b) harvesting the culture medium thus conditioned or lyse the        cell host, for example by sonication or by an osmotic shock;    -   c) separating or purifying, from the said culture medium, or        from the pellet of the resultant host cell lysate the thus        produced polypeptide of interest.    -   d) Optionally characterizing the produce polypeptide of        interest.

In a specific embodiment of the above method, step a) is preceded by astep wherein the nucleic acid coding for a CIDE B polypeptide, or afragment or a variant thereof, is inserted in an appropriate vector,optionally after an appropriate cleavage of this amplified nucleic acidwith one or several restriction endonucleases. The nucleic acid codingfor a CIDE B polypeptide or a fragment or a variant thereof may be theresulting product of an amplification reaction using a pair of primersaccording to the invention (by SDA, TAS, 3SR, NASBA, TMA etc.).

The polypeptides according to the invention may be characterized bybinding onto an immunoaffinity chromatography column on which polyclonalor monoclonal antibodies directed to a polypeptide of SEQ ID No 3, or afragment or a variant thereof, have previously been immobilized.

Purification of the recombinant proteins or peptides according to thepresent invention may be carried out by passage onto a Nickel or Cupperaffinity chromatography column. The Nickel chromatography column maycontain the Ni—NTA resin (Porath et al., 1975).

The polypeptides or peptides thus obtained may be purified, for exampleby high performance liquid chromatography, such as reverse phase and/orcationic exchange HPLC, as described by Rougeot et al. (1994). Thereason to prefer this kind of peptide or protein purification is thelack of byproducts found in the elution samples which renders theresultant purified protein or peptide more suitable for a therapeuticuse.

In a preferred embodiment, the CIDE B polypeptide comprises an aminoacid sequence of SEQ ID No 3 or a fragment or a variant thereof.

The CIDE B polypeptide of the amino acid sequence of SEQ ID No 3 has 219amino acids in length.

The human CIDE B protein presents 85% of identity with the murine CIDEB. This level of identity shows that the protein of the presentinvention is the human homologue of the murine CIDE B. In contrast, thehuman CIDE B protein of the invention presents only 42% of identity withthe human CIDE A protein.

The invention also encompasses a purified, isolated, or recombinantpolypeptides comprising an amino acid sequence having at least 90, 95,98 or 99% amino acid identity with the amino acid sequence of SEQ ID No3 or a fragment thereof.

In a preferred embodiment, the CIDE B polypeptide comprises an aminoacid sequence of SEQ ID No 3 or a fragment or a variant thereof. Thepresent invention also embodies isolated, purified, and recombinantpolypeptides comprising a contiguous span of at least 35, 40, 50, 100,150 or 200 amino acids of SEQ ID No 3. The present invention alsoembodies isolated, purified, and recombinant polypeptides comprising acontiguous span of at least 8 amino acids, preferably at least 10 aminoacids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or200 amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 1–29,47–70, 103–115, 124, 134, 169–185, and 203–219. Furthermore, the presentinvention embodies isolated, purified, and recombinant polypeptidescomprising a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 100, 150 or 200 amino acids of SEQ ID No 3, wherein saidcontiguous span includes at least 1, 2, 3, 5 or 10 of the followingamino acid positions: 7–11, 18–29, 47, 55–63, 70, 103–104, 111–115, 124,134, 169–173, 181–185, and 203–219.

Particular regions of the CIDE B polypeptide have interesting features.Two large hydrophilic and antigenic regions respectively begin at theamino acids in position 8 (N) and 79 (E), and respectively end at theamino acids in position 58 (E) and 146 (L) of the amino acid sequence ofCIDE B.

Four small regions having a good probability to be exposed to the outerenvironment are the amino acid sequences beginning at positions 31 (A),43 (H), 121 (G), and 138 (V) and respectively ending at positions 37(P), 47 (I), 128 (S) 144 (R) and of the CIDE B protein.

A further object of the present invention concerns a purified orisolated polypeptide which is encoded by a nucleic acid selected fromthe group consisting of SEQ ID Nos 1 and 2 or fragments or variantsthereof.

The invention also encompasses a purified or isolated nucleic acidencoding a CIDE B protein having the amino acid sequence of SEQ ID No 3,or a peptide fragment or variant thereof.

In a second preferred embodiment, a mutated CIDE B polypeptide comprisesamino acid changes with at least one amino acid deletion, substitutionor addition, preferably from 1 to 10, 20 or 30 amino acid deletions,substitutions or additions. The amino acid substitutions are generallynon conservative in terms of polarity, charge, hydrophilicity propertiesof the substitute amino acid when compared with the native amino acid.The amino acid changes occurring in such a mutated CIDE B polypeptidemay be determinant for the biological activity or for the capacity ofthe mutated CIDE B polypeptide to be recognized by antibodies raisedagainst a native CIDE B.

Such a mutated CIDE B protein may be the target of diagnostic tools,such as specific monoclonal or polyclonal antibodies, useful fordetecting the mutated CIDE B protein in a sample.

The invention also encompasses a CIDE B polypeptide or a fragment or avariant thereof in which at least one peptide bound has been modified asdescribed in the “Definitions” section.

Antibodies That Bind CIDE B Polypeptides of the Invention

Any CIDE B polypeptide or whole protein may be used to generateantibodies capable of specifically binding to an expressed CIDE Bprotein or fragments thereof as described.

One antibody composition of the invention is capable of specificallybinding or specifically bind to the variant of the CIDE B protein of SEQID No 3. For an antibody composition to specifically bind to CIDE B, itmust demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greaterbinding affinity for CIDE B protein than for another protein in anELISA, RIA, or other antibody-based binding assay.

In a preferred embodiment, the invention concerns antibody compositions,either polyclonal or monoclonal, capable of selectively binding, orselectively bind to an epitope containing a polypeptide comprising acontiguous span of at least 35, 40, 50, 100, 150 or 200 amino acids ofSEQ ID No 3; Optionally said epitope contains a polypeptide comprising acontiguous span of at least 8 amino acids, preferably at least 10 aminoacids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or200 amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 1–29,47–70, 103–115, 124, 134, 169–185, and 203–219; Optionally said epitopecontains a polypeptide comprising a contiguous span of at least 6 aminoacids, preferably at least 8 to 10 amino acids, more preferably at least12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 amino acids of SEQ ID No 3,wherein said contiguous span includes at least 1, 2, 3, 5 or 10 of thefollowing amino acid positions: 7–11, 18–29, 47, 55–63, 70, 103–104,111–115, 124, 134, 169–173, 181–185, and 203–219.

The invention also concerns a purified or isolated antibody capable ofspecifically binding to a mutated CIDE B protein or to a fragment orvariant thereof comprising an epitope of the mutated CIDE B protein. Inanother preferred embodiment, the present invention concerns an antibodycapable of binding to a polypeptide comprising at least 10 consecutiveamino acids of a CIDE B protein and including at least one of the aminoacids which can be encoded by the trait causing mutations.

In a preferred embodiment, the invention concerns the use in themanufacture of antibodies of a polypeptide comprising a contiguous spanof at least 35, 40, 50, 100, 150 or 200 amino acids of SEQ ID No 3;Optionally said polypeptide comprises a contiguous span of at least 8amino acids, preferably at least 10 amino acids, more preferably atleast 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 amino acids of SEQ IDNo 3, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe following amino acid positions: 1–29, 47–70, 103–115, 124, 134,169–185, and 203–219; Optionally said polypeptide comprises a contiguousspan of at least 6 amino acids, preferably at least 8 to 10 amino acids,more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 7–11,18–29, 47, 55–63, 70, 103–104, 111–115, 124, 134, 169–173, 181–185, and203–219.

The preferred fragments of CIDE B protein used for the preparation ofanti-human CIDE B antibodies comprise, or are comprised in, the aminoacid sequence located between the positions 8 and 58 of SEQ ID No 3 andbetween the positions 79 and 146 of SEQ ID No 3. More particularly, thefragments of CIDE B protein used for the preparation of anti-human CIDEB antibodies comprise the amino acid sequence located between thepositions 31 and 37, 43 and 47, 121 and 128, and 138 and 144 of thepolypeptide sequence of SEQ ID No 3.

The antibodies of the invention may be labeled by any one of theradioactive, fluorescent or enzymatic labels known in the art.

The CIDE B polypeptide of SEQ ID No 3 or a fragment thereof can be usedfor the preparation of polyclonal or monoclonal antibodies.

The CIDE B polypeptide expressed from a DNA sequence comprising at leastone of the nucleic acid sequences of SEQ ID Nos 1 and 2 may also be usedto generate antibodies capable of specifically binding to the CIDE Bpolypeptide of SEQ ID No 3 a fragment thereof.

The antibodies may be prepared from hybridomas according to thetechnique described by Kohler and Milstein in 1975. The polyclonalantibodies may be prepared by immunization of a mammal, especially amouse or a rabbit, with a polypeptide according to the invention that iscombined with an adjuvant of immunity, and then by purifying of thespecific antibodies contained in the serum of the immunized animal on aaffinity chromatography column on which has previously been immobilizedthe polypeptide that has been used as the antigen.

The present invention also includes, chimeric single chain Fv antibodyfragments (Martineau et al., 1998), antibody fragments obtained throughphage display libraries (Ridder et al., 1995; Vaughan et al., 1995) andhumanized antibodies (Reinmann et al., 1997; Leger et al., 1997).

Antibody preparations prepared according to either protocol are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body.

Consequently, the invention is also directed to a method for detectingspecifically the presence of a CIDE B polypeptide according to theinvention in a biological sample, said method comprising the followingsteps:

-   -   a) bringing into contact the biological sample with a polyclonal        or monoclonal antibody that specifically binds a CIDE B        polypeptide comprising an amino acid sequence of SEQ ID No 3, or        to a peptide fragment or variant thereof; and    -   b) detecting the antigen-antibody complex formed.

The invention also concerns a diagnostic kit for detecting in vitro thepresence of a CIDE B polypeptide according to the present invention in abiological sample, wherein said kit comprises:

-   -   a) a polyclonal or monoclonal antibody that specifically binds a        CIDE B polypeptide comprising an amino acid sequence of SEQ ID        No 3, or to a peptide fragment or variant thereof, optionally        labeled;    -   b) a reagent allowing the detection of the antigen-antibody        complexes formed, said reagent carrying optionally a label, or        being able to be recognized itself by a labeled reagent, more        particularly in the case when the above-mentioned monoclonal or        polyclonal antibody is not labeled by itself

Biallelic Markers of the CIDE B Gene

Identification of Biallelic Markers

There are two preferred methods through which the biallelic markers ofthe present invention can be generated. In a first method, DNA samplesfrom unrelated individuals are pooled together, following which thegenomic DNA of interest is amplified and sequenced. The nucleotidesequences thus obtained are then analyzed to identify significantpolymorphisms.

One of the major advantages of this method resides in the fact that thepooling of the DNA samples substantially reduces the number of DNAamplification reactions and sequencing which must be carried out.Moreover, this method is sufficiently sensitive so that a biallelicmarker obtained therewith usually shows a sufficient degree ofinformativeness for conducting association studies.

In a second method for generating biallelic markers, the DNA samples arenot pooled and are therefore amplified and sequenced individually. Theresulting nucleotide sequences obtained are then also analyzed toidentify significant polymorphisms.

It will readily be appreciated that when this second method is used, asubstantially higher number of DNA amplification reactions must becarried out. It will further be appreciated that including suchpotentially less informative biallelic markers in association studies toidentify potential genetic associations with a trait may allow in somecases the direct identification of causal mutations, which may,depending on their penetrance, be rare mutations. This method is usuallypreferred when biallelic markers need to be identified in order toperform association studies within candidate genes.

In both methods, the genomic DNA samples from which the biallelicmarkers of the present invention are generated are preferably obtainedfrom unrelated individuals corresponding to a heterogeneous populationof known ethnic background, or from familial cases.

The number of individuals from whom DNA samples are obtained can varysubstantially, preferably from about 10 to about 1000, preferably fromabout 50 to about 200 individuals. It is usually preferred to collectDNA samples from at least about 100 individuals in order to havesufficient polymorphic diversity in a given population to generate asmany markers as possible and to generate statistically significantresults.

As for the source of the genomic DNA to be subjected to analysis, anytest sample can be foreseen without any particular limitation. Thepreferred source of genomic DNA used in the context of the presentinvention is the peripheral venous blood of each donor.

The techniques of DNA extraction are well-known to the skilledtechnician. Details of a preferred embodiment are provided in Example 1.

DNA samples can be pooled or unpooled for the amplification step. DNAamplification techniques are well-known to those skilled in the art. ThePCR technology is the preferred amplification technique used in thepresent invention. A typical example of a PCR reaction suitable for thepurposes of the present invention is provided in Example 2.

The primers used for the amplification are as defined above. Preferredprimers of the invention include the nucleotide sequences of B(12-73),B(12-74), C(12-73), C(12-74), D(12-73-49), D(12-74-38), E(12-73-49), andE(12-74-38). More preferred primers of the invention include thenucleotide sequences of B(12-73), B(12-74), C(12-73), and C(12-74).

The amplification products generated as described above with the primersof the invention are then sequenced using methods known and available tothe skilled technician. Preferably, the amplified DNA is subjected toautomated dideoxy terminator sequencing reactions using a dye-primercycle sequencing protocol. Following gel image analysis and DNA sequenceextraction, sequence data are automatically processed with adequatesoftware to assess sequence quality.

The presence of biallelic sites are detected among individual or pooledamplified fragment sequences. Polymorphism search is based on thepresence of superimposed peaks in the electrophoresis pattern. Thesepeaks which present distinct colors correspond to two differentnucleotides at the same position on the sequence. The polymorphism hasto be detected on both strands for validation.

The biallelic markers of the present invention are disclosed in Table 2of Example 3. Their location on the CIDE B gene is indicated as featuresin SEQ ID No 1. The pair of amplification primers are listed in thesequence listing in features of the SEQ ID No 1 and are described inTable 1 of example 2, these primers allowing the amplification of anucleic acid containing the polymorphic base that defines this biallelicmarker.

In the present invention, the biallelic markers can be defined bynucleotide sequences corresponding to oligonucleotides of 47 bases inlength comprising at the middle one of the polymorphic base. Moreparticularly, the biallelic markers can be defined by thepolynucleotides P(12-73-49) and P(12-74-38).

The biallelic markers 12-73-49 and 12-74-38 are located in the 3′regulatory region and form part of the present invention.

The biallelic markers contained in the human CIDE B gene are usefultools to perform association studies between the statisticallysignificant occurrence of an allele of said biallelic marker in thegenome of an individual and a specific phenotype, including a phenotypeconsisting of a disorder related to apoptosis such as cancer or AIDS.The biallelic markers of the invention can also be used, for example,for the generation of genetic map, the linkage analysis.

Genotyping of Biallelic Markers

Any method known in the art can be used to identify the nucleotidepresent at a biallelic marker site. Since the biallelic marker allele tobe detected has been identified and specified in the present invention,detection will prove simple for one of ordinary skill in the art byemploying any of a number of techniques. Many genotyping methods requirethe previous amplification of the DNA region carrying the biallelicmarker of interest. While the amplification of target or signal is oftenpreferred at present, ultrasensitive detection methods which do notrequire amplification are also encompassed by the present genotypingmethods. Methods well-known to those skilled in the art that can be usedto detect biallelic polymorphisms include methods such as, conventionaldot blot analyzes, single strand conformational polymorphism analysis(SSCP) described by Orita et al.(1989), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis, mismatch cleavagedetection, and other conventional techniques as described in Sheffieldet al.(1991), White et al.(1992), Grompe et al.(1989 and 1993). Anothermethod for determining the identity of the nucleotide present at aparticular polymorphic site employs a specialized exonuclease-resistantnucleotide derivative as described in U.S. Pat. No. 4,656,127.

Preferred methods involve directly determining the identity of thenucleotide present at a biallelic marker site by sequencing assay,enzyme-based mismatch detection assay, or hybridization assay. Thefollowing is a description of some preferred methods. A highly preferredmethod is the microsequencing technique. The term “sequencing” isgenerally used herein to refer to polymerase extension of duplexprimer/template complexes and includes both traditional sequencing andmicrosequencing.

1) Sequencing Assays

The nucleotide present at a polymorphic site can be determined bysequencing methods. In a preferred embodiment, DNA samples are subjectedto PCR amplification before sequencing as described above.

Preferably, the amplified DNA is subjected to automated dideoxyterminator sequencing reactions using a dye-primer cycle sequencingprotocol. Sequence analysis allows the identification of the basepresent at the biallelic marker site.

2) Microsequencing Assays

In microsequencing methods, the nucleotide at a polymorphic site in atarget DNA is detected by a single nucleotide primer extension reaction.This method involves appropriate microsequencing primers which,hybridize just upstream of the polymorphic base of interest in thetarget nucleic acid. A polymerase is used to specifically extend the 3′end of the primer with one single ddNTP (chain terminator) complementaryto the nucleotide at the polymorphic site. Next the identity of theincorporated nucleotide is determined in any suitable way.

Typically, microsequencing reactions are carried out using fluorescentddNTPs and the extended microsequencing primers are analyzed byelectrophoresis on ABI 377 sequencing machines to determine the identityof the incorporated nucleotide as described in EP 412 883. Alternativelycapillary electrophoresis can be used in order to process a highernumber of assays simultaneously. An example of a typical microsequencingprocedure that can be used in the context of the present invention isprovided in Example 4.

Different approaches can be used for the labeling and detection ofddNTPs. A homogeneous phase detection method based on fluorescenceresonance energy transfer has been described by Chen and Kwok (1997) andChen et al.(1997). In this method, amplified genomic DNA fragmentscontaining polymorphic sites are incubated with a 5′-fluorescein-labeledprimer in the presence of allelic dye-labeled dideoxyribonucleosidetriphosphates and a modified Taq polymerase. The dye-labeled primer isextended one base by the dye-terminator specific for the allele presenton the template. At the end of the genotyping reaction, the fluorescenceintensities of the two dyes in the reaction mixture are analyzeddirectly without separation or purification. All these steps can beperformed in the same tube and the fluorescence changes can be monitoredin real time. Alternatively, the extended primer may be analyzed byMALDI-TOF Mass Spectrometry. The base at the polymorphic site isidentified by the mass added onto the microsequencing primer (see Haffand Smirnov, 1997).

Microsequencing may be achieved by the established microsequencingmethod or by developments or derivatives thereof. Alternative methodsinclude several solid-phase microsequencing techniques. The basicmicrosequencing protocol is the same as described previously, exceptthat the method is conducted as a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized or captured onto asolid support. To simplify the primer separation and the terminalnucleotide addition analysis, oligonucleotides are attached to solidsupports or are modified in such ways that permit affinity separation aswell as polymerase extension. The 5′ ends and internal nucleotides ofsynthetic oligonucleotides can be modified in a number of different waysto permit different affinity separation approaches, e.g., biotinylation.If a single affinity group is used on the oligonucleotides, theoligonucleotides can be separated from the incorporated terminatorregent. This eliminates the need of physical or size separation. Morethan one oligonucleotide can be separated from the terminator reagentand analyzed simultaneously if more than one affinity group is used.This permits the analysis of several nucleic acid species or morenucleic acid sequence information per extension reaction. The affinitygroup need not be on the priming oligonucleotide but could alternativelybe present on the template. For example, immobilization can be carriedout via an interaction between biotinylated DNA and streptavidin-coatedmicrotitration wells or avidin-coated polystyrene particles. In the samemanner, oligonucleotides or templates may be attached to a solid supportin a high-density format. In such solid phase microsequencing reactions,incorporated ddNTPs can be radiolabeled (Syvänen, 1994) or linked tofluorescein (Livak and Hainer, 1994). The detection of radiolabeledddNTPs can be achieved through scintillation-based techniques. Thedetection of fluorescein-linked ddNTPs can be based on the binding ofantifluorescein antibody conjugated with alkaline phosphatase, followedby incubation with a chromogenic substrate (such as p-nitrophenylphosphate). Other possible reporter-detection pairs include: ddNTPlinked to dinitrophenyl (DNP) and anti-DNP alkaline phosphataseconjugate (Harju et al., 1993) or biotinylated ddNTP and horseradishperoxidase-conjugated streptavidin with o-phenylenediamine as asubstrate (WO 92/15712). As yet another alternative solid-phasemicrosequencing procedure, Nyren et al.(1993) described a method relyingon the detection of DNA polymerase activity by an enzymatic luminometricinorganic pyrophosphate detection assay (ELIDA).

Pastinen et al.(1997) describe a method for multiplex detection ofsingle nucleotide polymorphism in which the solid phase minisequencingprinciple is applied to an oligonucleotide array format. High-densityarrays of DNA probes attached to a solid support (DNA chips) are furtherdescribed below.

In one aspect the present invention provides polynucleotides and methodsto genotype one or more biallelic markers of the present invention byperforming a microsequencing assay. Preferred microsequencing primersinclude the nucleotide sequences D(12-73-49), D(12-74-38), E(12-73-49),and E(12-74-38). It will be appreciated that the microsequencing primerslisted in Example 4 are merely exemplary and that, any primer having a3′ end immediately adjacent to the polymorphic nucleotide may be used.Similarly, it will be appreciated that microsequencing analysis may beperformed for any biallelic marker or any combination of biallelicmarkers of the present invention. One aspect of the present invention isa solid support which includes one or more microsequencing primerslisted in Example 4, or fragments comprising at least 8, 12, 15, 20, 25,30, 40, or 50 consecutive nucleotides thereof, to the extent that suchlengths are consistent with the primer described, and having a 3′terminus immediately upstream of the corresponding biallelic marker, fordetermining the identity of a nucleotide at a biallelic marker site.

3) Mismatch Detection Assays Based on Polymerases and Ligases

In one aspect the present invention provides polynucleotides and methodsto determine the allele of one or more biallelic markers of the presentinvention in a biological sample, by mismatch detection assays based onpolymerases and/or ligases. These assays are based on the specificity ofpolymerases and ligases. Polymerization reactions places particularlystringent requirements on correct base pairing of the 3′ end of theamplification primer and the joining of two oligonucleotides hybridizedto a target DNA sequence is quite sensitive to mismatches close to theligation site, especially at the 3′ end. Methods, primers and variousparameters to amplify DNA fragments comprising biallelic markers of thepresent invention are further described above in “Amplification Of DNAFragments Comprising Biallelic Markers”.

Allele Specific Amplification Primers

Discrimination between the two alleles of a biallelic marker can also beachieved by allele specific amplification, a selective strategy, wherebyone of the alleles is amplified without amplification of the otherallele. For allele specific amplification, at least one member of thepair of primers is sufficiently complementary with a region of a CIDE Bgene comprising the polymorphic base of a biallelic marker of thepresent invention to hybridize therewith and to initiate theamplification. Such primers are able to discriminate between the twoalleles of a biallelic marker.

This is accomplished by placing the polymorphic base at the 3′ end ofone of the amplification primers. Because the extension forms from the3′ end of the primer, a mismatch at or near this position has aninhibitory effect on amplification. Therefore, under appropriateamplification conditions, these primers only direct amplification ontheir complementary allele. Determining the precise location of themismatch and the corresponding assay conditions are well within theordinary skill in the art.

Ligation/Amplification Based Methods

The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotideswhich are designed to be capable of hybridizing to abutting sequences ofa single strand of a target molecules. One of the oligonucleotides isbiotinylated, and the other is detectably labeled. If the precisecomplementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate that can be captured and detected. OLA is capableof detecting single nucleotide polymorphisms and may be advantageouslycombined with PCR as described by Nickerson et al.(1990). In thismethod, PCR is used to achieve the exponential amplification of targetDNA, which is then detected using OLA.

Other amplification methods which are particularly suited for thedetection of single nucleotide polymorphism include LCR (ligase chainreaction), Gap LCR (GLCR) which are described above in “DNAAmplification”. LCR uses two pairs of probes to exponentially amplify aspecific target. The sequences of each pair of oligonucleotides, isselected to permit the pair to hybridize to abutting sequences of thesame strand of the target. Such hybridization forms a substrate for atemplate-dependant ligase. In accordance with the present invention, LCRcan be performed with oligonucleotides having the proximal and distalsequences of the same strand of a biallelic marker site. In oneembodiment, either oligonucleotide will be designed to include thebiallelic marker site. In such an embodiment, the reaction conditionsare selected such that the oligonucleotides can be ligated together onlyif the target molecule either contains or lacks the specific nucleotidethat is complementary to the biallelic marker on the oligonucleotide. Inan alternative embodiment, the oligonucleotides will not include thebiallelic marker, such that when they hybridize to the target molecule,a “gap” is created as described in WO 90/01069. This gap is then“filled” with complementary dNTPs (as mediated by DNA polymerase), or byan additional pair of oligonucleotides. Thus at the end of each cycle,each single strand has a complement capable of serving as a targetduring the next cycle and exponential allele-specific amplification ofthe desired sequence is obtained.

Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method fordetermining the identity of a nucleotide at a preselected site in anucleic acid molecule (WO 95/21271). This method involves theincorporation of a nucleoside triphosphate that is complementary to thenucleotide present at the preselected site onto the terminus of a primermolecule, and their subsequent ligation to a second oligonucleotide. Thereaction is monitored by detecting a specific label attached to thereaction's solid phase or by detection in solution.

4) Hybridization Assay Methods

A preferred method of determining the identity of the nucleotide presentat a biallelic marker site involves nucleic acid hybridization. Thehybridization probes, which can be conveniently used in such reactions,preferably include the probes defined herein. Any hybridization assaymay be used including Southern hybridization, Northern hybridization,dot blot hybridization and solid-phase hybridization (see Sambrook etal., 1989).

Hybridization refers to the formation of a duplex structure by twosingle stranded nucleic acids due to complementary base pairing.Hybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch. Specific probes can be designed that hybridize to one form ofa biallelic marker and not to the other and therefore are able todiscriminate between different allelic forms. Allele-specific probes areoften used in pairs, one member of a pair showing perfect match to atarget sequence containing the original allele and the other showing aperfect match to the target sequence containing the alternative allele.Hybridization conditions should be sufficiently stringent that there isa significant difference in hybridization intensity between alleles, andpreferably an essentially binary response, whereby a probe hybridizes toonly one of the alleles. Stringent, sequence specific hybridizationconditions, under which a probe will hybridize only to the exactlycomplementary target sequence are well known in the art (Sambrook etal., 1989). Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. Althoughsuch hybridization can be performed in solution, it is preferred toemploy a solid-phase hybridization assay. The target DNA comprising abiallelic marker of the present invention may be amplified prior to thehybridization reaction. The presence of a specific allele in the sampleis determined by detecting the presence or the absence of stable hybridduplexes formed between the probe and the target DNA. The detection ofhybrid duplexes can be carried out by a number of methods. Variousdetection assay formats are well known which utilize detectable labelsbound to either the target or the probe to enable detection of thehybrid duplexes. Typically, hybridization duplexes are separated fromunhybridized nucleic acids and the labels bound to the duplexes are thendetected. Those skilled in the art will recognize that wash steps may beemployed to wash away excess target DNA or probe as well as unboundconjugate. Further, standard heterogeneous assay formats are suitablefor detecting the hybrids using the labels present on the primers andprobes.

Two recently developed assays allow hybridization-based allelediscrimination with no need for separations or washes (see Landegren U.et al., 1998). The TaqMan assay takes advantage of the 5′ nucleaseactivity of Taq DNA polymerase to digest a DNA probe annealedspecifically to the accumulating amplification product. TaqMan probesare labeled with a donor-acceptor dye pair that interacts viafluorescence energy transfer. Cleavage of the TaqMan probe by theadvancing polymerase during amplification dissociates the donor dye fromthe quenching acceptor dye, greatly increasing the donor fluorescence.All reagents necessary to detect two allelic variants can be assembledat the beginning of the reaction and the results are monitored in realtime (see Livak et al., 1995). In an alternative homogeneoushybridization based procedure, molecular beacons are used for allelediscriminations. Molecular beacons are hairpin-shaped oligonucleotideprobes that report the presence of specific nucleic acids in homogeneoussolutions. When they bind to their targets they undergo a conformationalreorganization that restores the fluorescence of an internally quenchedfluorophore (Tyagi et al., 1998).

The polynucleotides provided herein can be used to produce probes whichcan be used in hybridization assays for the detection of biallelicmarker alleles in biological samples. These probes are characterized inthat they preferably comprise between 8 and 50 nucleotides, and in thatthey are sufficiently complementary to a sequence comprising a biallelicmarker of the present invention to hybridize thereto and preferablysufficiently specific to be able to discriminate the targeted sequencefor only one nucleotide variation. A particularly preferred probe is 25nucleotides in length. Preferably the biallelic marker is within 4nucleotides of the center of the polynucleotide probe. In particularlypreferred probes, the biallelic marker is at the center of saidpolynucleotide. Preferred probes comprise a nucleotide sequence selectedfrom the group consisting of amplicons listed in Table 1 and thesequences complementary thereto, or a fragment thereof, said fragmentcomprising at least about 8 consecutive nucleotides, preferably 10, 15,20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides andcontaining a polymorphic base. Preferred probes comprise a nucleotidesequence selected from the group consisting of P(12-73-49) andP(12-74–38) and the sequences complementary thereto. In preferredembodiments the polymorphic base(s) are within 5, 4, 3, 2, 1,nucleotides of the center of the said polynucleotide, more preferably atthe center of said polynucleotide.

Preferably the probes of the present invention are labeled orimmobilized on a solid support. Labels and solid supports are furtherdescribed in “Oligonucleotide Probes and Primers”. The probes can benon-extendable as described in “Oligonucleotide Probes and Primers”.

By assaying the hybridization to an allele specific probe, one candetect the presence or absence of a biallelic marker allele in a givensample. High-Throughput parallel hybridization in array format isspecifically encompassed within “hybridization assays” and are describedbelow.

5) Hybridization to Addressable Arrays of Oligonucleotides

Hybridization assays based on oligonucleotide arrays rely on thedifferences in hybridization stability of short oligonucleotides toperfectly matched and mismatched target sequence variants. Efficientaccess to polymorphism information is obtained through a basic structurecomprising high-density arrays of oligonucleotide probes attached to asolid support (e.g., the chip) at selected positions. Each DNA chip cancontain thousands to millions of individual synthetic DNA probesarranged in a grid-like pattern and miniaturized to the size of a dime.

The chip technology has already been applied with success in numerouscases. For example, the screening of mutations has been undertaken inthe BRCA1 gene, in S. cerevisiae mutant strains, and in the proteasegene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al., 1996; Kozalet al., 1996). Chips of various formats for use in detecting biallelicpolymorphisms can be produced on a customized basis by Affymetrix(GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.

In general, these methods employ arrays of oligonucleotide probes thatare complementary to target nucleic acid sequence segments from anindividual which, target sequences include a polymorphic marker. EP785280 describes a tiling strategy for the detection of singlenucleotide polymorphisms. Briefly, arrays may generally be “tiled” for alarge number of specific polymorphisms. By “tiling” is generally meantthe synthesis of a defined set of oligonucleotide probes which is madeup of a sequence complementary to the target sequence of interest, aswell as preselected variations of that sequence, e.g., substitution ofone or more given positions with one or more members of the basis set ofnucleotides. Tiling strategies are further described in PCT applicationNo. WO 95/11995. In a particular aspect, arrays are tiled for a numberof specific, identified biallelic marker sequences. In particular, thearray is tiled to include a number of detection blocks, each detectionblock being specific for a specific biallelic marker or a set ofbiallelic markers. For example, a detection block may be tiled toinclude a number of probes, which span the sequence segment thatincludes a specific polymorphism. To ensure probes that arecomplementary to each allele, the probes are synthesized in pairsdiffering at the biallelic marker. In addition to the probes differingat the polymorphic base, monosubstituted probes are also generally tiledwithin the detection block. These monosubstituted probes have bases atand up to a certain number of bases in either direction from thepolymorphism, substituted with the remaining nucleotides (selected fromA, T, G, C and U). Typically the probes in a tiled detection block willinclude substitutions of the sequence positions up to and includingthose that are 5 bases away from the biallelic marker. Themonosubstituted probes provide internal controls for the tiled array, todistinguish actual hybridization from artefactual cross-hybridization.Upon completion of hybridization with the target sequence and washing ofthe array, the array is scanned to determine the position on the arrayto which the target sequence hybridizes. The hybridization data from thescanned array is then analyzed to identify which allele or alleles ofthe biallelic marker are present in the sample. Hybridization andscanning may be carried out as described in PCT application No. WO92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186.

Thus, in some embodiments, the chips may comprise an array of nucleicacid sequences of fragments of about 15 nucleotides in length. Infurther embodiments, the chip may comprise an array including at leastone of the sequences selected from the group consisting of ampliconslisted in table 1 and the sequences complementary thereto, or a fragmentthereof, said fragment comprising at least about 8 consecutivenucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or50 consecutive nucleotides and containing a polymorphic base. Inpreferred embodiments the polymorphic base is within 5, 4, 3, 2, 1,nucleotides of the center of the said polynucleotide, more preferably atthe center of said polynucleotide. In some embodiments, the chip maycomprise an array of at least 2, 3, 4, 5,6, 7, 8 or more of thesepolynucleotides of the invention. Solid supports and polynucleotides ofthe present invention attached to solid supports are further describedin “Oligonucleotide Probes And Primers”.

6) Integrated Systems

Another technique, which may be used to analyze polymorphisms, includesmulticomponent integrated systems, which miniaturize andcompartmentalize processes such as PCR and capillary electrophoresisreactions in a single functional device. An example of such technique isdisclosed in U.S. Pat. No. 5,589,136, which describes the integration ofPCR amplification and capillary electrophoresis in chips.

Integrated systems can be envisaged mainly when microfluidic systems areused. These systems comprise a pattern of microchannels designed onto aglass, silicon, quartz, or plastic wafer included on a microchip. Themovements of the samples are controlled by electric, electroosmotic orhydrostatic forces applied across different areas of the microchip tocreate functional microscopic valves and pumps with no moving parts.

For genotyping biallelic markers, the microfluidic system may integratenucleic acid amplification, microsequencing, capillary electrophoresisand a detection method such as laser-induced fluorescence detection.

Expression of a Regulatory or Coding Polynucleotide of CIDE B.

Any of the regulatory polynucleotides or the coding polynucleotides ofthe invention may be inserted into recombinant vectors for expression ina recombinant host cell or a recombinant host organism.

Thus, the present invention also encompasses a family of recombinantvectors that contains either a regulatory polynucleotide selected fromthe group consisting of any one of the regulatory polynucleotidesderived from the CIDE B genomic sequence, or a coding polynucleotidefrom the CIDE B genomic sequence. Consequently, the present inventionfurther deals with a recombinant vector comprising either a regulatorypolynucleotide contained in the nucleic acid of SEQ ID No 1, or apolynucleotide comprising the CIDE B coding sequence, or both.

In a first preferred embodiment, a recombinant vector of the inventionis used as an expression vector; (a) the CIDE B regulatory sequencecomprised therein drives the expression of a coding polynucleotideoperably linked thereto; (b) the CIDE B coding sequence is operablylinked to regulation sequences allowing its expression in a suitablecell host and/or host organism.

In a second preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide derived from a CIDE Bgenomic sequence selected from the group consisting of the nucleic acidsof SEQ ID No 1 or a CIDE B cDNA in a suitable cell host, thispolynucleotide being amplified at every time that the recombinant vectorreplicates.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding a CIDE B protein, preferably theCIDE B protein of the amino acid sequence of SEQ ID No 3 or variants orfragments thereof, under the control of a regulatory sequence selectedamong the CIDE B regulatory polynucleotides, or alternatively under thecontrol of an exogenous regulatory sequence.

A recombinant expression vector comprising a nucleic acid selected fromthe group consisting of the 5′ regulatory region, or biologically activefragments or variants thereof, is also part of the present invention.

Generally, a recombinant vector of the invention may comprise any of thepolynucleotides described herein, including regulatory sequences, andcoding sequences, as well as any CIDE B primer or probe as definedabove. More particularly, the recombinant vectors of the presentinvention can comprise any of the polynucleotides described in the “CIDEB cDNA Sequences” section, the “Coding Regions of CIDE B” section,“Genomic sequence of CIDE B” section and the “Oligonucleotide Probes AndPrimers” section.

Some of the elements which can be found in the vectors of the presentinvention are described in further detail in the following sections.

Vectors

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or evena linear DNA molecule which may consist of a chromosomal,non-chromosomal and synthetic DNA. Such a recombinant vector cancomprise a transcriptional unit comprising an assembly of:

-   -   (1) a genetic element or elements having a regulatory role in        gene expression, for example promoters or enhancers. Enhancers        are cis-acting elements of DNA, usually from about 10 to 300 bp        in length that act on the promoter to increase the        transcription.    -   (2) a structural or coding sequence which is transcribed into        mRNA and eventually translated into a polypeptide, and    -   (3) appropriate transcription initiation and termination        sequences. Structural units intended for use in yeast or        eukaryotic expression systems preferably include a leader        sequence enabling extracellular secretion of translated protein        by a host cell. Alternatively, where a recombinant protein is        expressed without a leader or transport sequence, it may include        an N-terminal residue. This residue may or may not be        subsequently cleaved from the expressed recombinant protein to        provide a final product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium.

The selectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. coli, or levan saccharase for mycobacteria.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223–3 (Pharmacia, Uppsala, Sweden), andGEM1 (Promega Biotec, Madison, WI, USA).

Large numbers of suitable vectors and promoters are known to those ofskill in the art, and commercially available, such as bacterial vectors:pQE70, pQE60, pQE9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223–3, pKK233–3, pDR540, pRIT5 (Pharmacia); or eukaryoticvectors : pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV,pMSG, pSVL (Pharmacia); baculovirus transfer vector pVL1392/1393(Pharmingen); pQE-30 (QIAexpress).

A suitable vector for the expression of the CIDE B polypeptide of SEQ IDNo 3 or fragments or variants thereof is a baculovirus vector that canbe propagated in insect cells and in insect cell lines. A specificsuitable host vector system is the pVL1392/1393 baculovirus transfervector (Pharmingen) that is used to transfect the SF9 cell line (ATCCN^(o)CRL 1711) which is derived from Spodoptera frugiperda.

Other suitable vectors for the expression of the CIDE B polypeptide ofSEQ ID No 3 or fragments or variants thereof in a baculovirus expressionsystem include those described by Chai et al. (1993), Vlasak et al.(1983) and Lenhard et al. (1996).

Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′-flanking non-transcribedsequences. DNA sequences derived from the SV40 viral genome, for exampleSV40 origin, early promoter, enhancer, splice and polyadenylation sitesmay be used to provide the required non-transcribed genetic elements.

Promoters

The suitable promoter regions used in the expression vectors accordingto the present invention are chosen taking into account the cell host inwhich the heterologous gene has to be expressed.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted.

Preferred bacterial promoters are the LacI, LacZ, the T3 or T7bacteriophage RNA polymerase promoters, the polyhedrin promoter, or thep10 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983;O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.

Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors. Particularly preferred bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryoticpromoters include CMV immediate early, HSV thymidine kinase, early andlate SV40, LTRs from retrovirus, and mouse metallothionein-L. Selectionof a convenient vector and promoter is well within the level of ordinaryskill in the art.

The choice of a promoter is well within the ability of a person skilledin the field of genetic egineering. For example, one may refer to thebook of Sambrook et al. (1989) or also to the procedures described byFuller et al. (1996).

The vector containing the appropriate DNA sequence as described above,more preferably CIDE B gene regulatory polynucleotide, a polynucleotideencoding the CIDE B polypeptide of SEQ ID No 3 or both of them, can beutilized to transform an appropriate host to allow the expression of thedesired polypeptide or polynucleotide.

Other Types of Vectors

The in vivo expression of a CIDE B polypeptide of SEQ ID No No 3 orfragments or variants thereof may be useful in order to correct agenetic defect related to the expression of the native gene in a hostorganism or to the production of a biologically inactive CIDE B protein.

Consequently, the present invention also deals with recombinantexpression vectors mainly designed for the in vivo production of theCIDE B polypeptide of SEQ ID No No 3 or fragments or variants thereof bythe introduction of the appropriate genetic material in the organism ofthe patient to be treated. This genetic material may be introduced invitro in a cell that has been previously extracted from the organism,the modified cell being subsequently reintroduced in the said organism,directly in vivo into the appropriate tissue.

By <<vector>> according to this specific embodiment of the invention isintended either a circular or a linear DNA molecule.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect.

In a specific embodiment, the invention provides a composition for thein vivo production of the CIDE B protein or polypeptide describedherein. It comprises a naked polynucleotide operatively coding for thispolypeptide, in solution in a physiologically acceptable carrier, andsuitable for introduction into a tissue to cause cells of the tissue toexpress the said protein or polypeptide.

Compositions comprising a polynucleotide are described in PCTapplication N^(o) WO 90/11092 (Vical Inc.) and also in PCT applicationN^(o) WO 95/11307 (Institut Pasteur, INSERM, Universitéd'Ottawa) as wellas in the articles of Tacson et al. (1996) and of Huygen et al. (1996).

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0,1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it maybe introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired CIDE Bpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg (1996) or Ohno et al. (1994). Anotherpreferred recombinant adenovirus according to this specific embodimentof the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad5) or an adenovirus of animal origin ( French patent application N^(o)FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vitro gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., 1989, Julan et al., 1992 andNeda et al., 1991.

Yet another viral vector system that is contemplated by the inventionconsists in the adeno-associated virus (AAV). The adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al., 1992). It isalso one of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (Flotte etal., 1992; Samulski et al., 1989; McLaughlin et al., 1989). Oneadvantageous feature of AAV derives from its reduced efficacy fortransducing primary cells relative to transformed cells.

Other compositions containing a vector of the invention advantageouslycomprise an oligonucleotide fragment of the nucleic sequence SEQ ID No2, preferably a fragment including the start codon of the CIDE B gene,as an antisense tool that inhibits the expression of the correspondingCIDE B gene. Preferred methods using antisense polynucleotide accordingto the present invention are the procedures described by Sczakiel et al.(1995) or those described in PCT Application No WO 95/24223.

Preferably, the antisense tools are chosen among the polynucleotides(15–200 bp long) that are complementary to the 5′end of the CIDE B mRNA.In another embodiment, a combination of different antisensepolynucleotides complementary to different parts of the desired targetedgene are used.

Preferred antisense polynucleotides according to the present inventionare complementary to a sequence of the mRNAs of CIDE B that contains thetranslation initiation codon ATG.

Host Cells

Another object of the invention consists in host cell that have beentransformed or transfected with one of the polynucleotides describedtherein, and more precisely a polynucleotide either comprising a CIDE Bregulatory polynucleotide or the coding sequence of the CIDE Bpolypeptide having the amino acid sequence of SEQ ID No 3 or fragmentsor variants thereof. Are included host cells that are transformed(prokaryotic cells) or that are transfected (eukaryotic cells) with arecombinant vector such as one of those described above.

A recombinant host cell of the invention comprises any one of thepolynucleotides or the recombinant vectors described therein. Moreparticularly, the cell hosts of the present invention can comprise anyof the polynucleotides described in “CIDEB cDNA Sequences” section, the“Coding Regions Of CIDE B” section, “Genomic sequence of CIDE B”section, the “Oligonucleotide Probes And Primers” section and the“Vectors for the expression of a regulatory or coding polynucleotide ofCIDE B” section.

Preferred host cells used as recipients for the expression vectors ofthe invention are the following:

-   -   a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-α        strain) or Bacillus subtilis.    -   b) Eukaryotic host cells : HeLa cells (ATCC N^(o)CCL2;        N^(o)CCL2.1; N^(o)CCL2.2), Cv 1 cells (ATCC N^(o)CCL70), COS        cells (ATCC N^(o)CRL1650; N^(o)CRL1651), Sf-9 cells (ATCC        N^(o)CRL1711).

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skill artisan.

Transgenic Animals

The terms “transgenic animals” or “host animals” are used herein todesignate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. Preferred animals are non-human mammals and include thosebelonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats)and Oryctogalus (e.g. rabbits) which have their genome artificially andgenetically altered by the insertion of a nucleic acid according to theinvention.

The transgenic animals of the invention all include within a pluralityof their cells a cloned recombinant or synthetic DNA sequence, morespecifically one of the purified or isolated nucleic acids comprising aCIDE B coding sequence, a CIDE B regulatory polynucleotide or a DNAsequence encoding an antisense polynucleotide such as described in thepresent specification.

More particularly, transgenic animals according to the invention containin their somatic cells and/or in their germ line cells any of thepolynucleotides described in “CIDE B cDNA Sequences” section, the“Coding Regions Of CIDE B” section, “Genomic sequence of CIDE B”section, the “Oligonucleotide Probes And Primers” section and the“Vectors for the expression of a regulatory or coding polynucleotide ofCIDE B” section.

The replacement of the native genomic CIDE B sequence by a defectivecopy of said sequence may be preformed by techniques of gene targeting.Such techniques are notably described by Burright et al. (1997), Bateset al. (1997), Mangiarini et al. (1997), Davies et al. (1997).

Second preferred transgenic animals of the invention have the murineCIDE B gene replaced either by a defective copy of the murine CIDE Bgene or by an interrupted copy of the human CIDE B gene. A “defectivecopy” of a murine or a human CIDE B gene, is intended to designate amodified copy of these genes that is not or poorly transcribed in theresulting recombinant host animal or a modified copy of these genesleading to the absence of synthesis of the corresponding translationproduct or alternatively leading to a modified and/or truncatedtranslation product lacking the biological activity of the wild typeCIDE B protein. The altered translation product thus contains amino acidmodifications, deletions and substitutions. Modifications and deletionsmay render the naturally occurring gene nonfunctional, thus leading to a“knockout animal”. These transgenic animals are critical for thecreation of animal models of human diseases, and for eventual treatmentof disorders related to apoptosis such as cancer or AIDS. Examples ofsuch knockout mice are described in the PCT Applications Nos WO97/34641, WO 96/12792 and WO 98/02354.

The endogenous murine CIDE B gene can be interrupted by the insertion,between two contiguous nucleotide of said gene, of a part of all of amarker gene placed under the control of the appropriate promoter, forexample the endogenous promoter of the endogenous murine CIDE B gene.The marker gene may be the neomycin resistance gene (neo) that may beoperably linked to the phosphoglycerate kinase-1 (PGK-1) promoter, asdescribed in the PCT Application No WO 98/02534.

Thus, the invention is also directed to a transgenic animal contain intheir somatic cells and/or in their germ line cells a polynucleotideselected from the following group of polynucleotides:

-   -   a) a defective copy of the human CIDE B gene;    -   b) a defective copy of the endogenous CIDE B gene, wherein the        expression “endogenous CIDE B gene” designates a CIDE B gene        that is naturally present within the genome of the animal host        to be genetically modified.

The invention also concerns a method for obtaining transgenic animals,wherein said methods comprise the steps of:

-   -   a) replacing the endogenous copy of the animal CIDE B gene by a        nucleic acid selected from the group consisting of a defective        copy of the human CIDE B gene and a defective copy of the        endogenous CIDE B gene in animal cells, preferably embryonic        stem cells (ES);    -   b) introducing the recombinant animal cells obtained at step a)        in embryos, notably blastocysts of the animal;    -   c) selecting the resulting transgenic animals, for example by        detecting the defective copy of a CIDE B gene with one or        several primers or probes according to the invention.

Optionally, the transgenic animals may be bred together in order toobtain homozygous transgenic animals for the defective copy of the CIDEB gene introduced.

The transgenic animals of the invention thus contain specific sequencesof exogenous genetic material such as the nucleotide sequences describedabove in detail.

In a first preferred embodiment, these transgenic animals may be goodexperimental models in order to study the diverse pathologies related todisorders associated to apoptosis, in particular concerning thetransgenic animals within the genome of which has been inserted one orseveral copies of a polynucleotide encoding a native CIDE B protein, oralternatively a mutant CIDE B protein.

In a second preferred embodiment, these transgenic animals may express adesired polypeptide of interest under the control of the regulatorypolynucleotides of the CIDE B gene, leading to good yields in thesynthesis of this protein of interest, and eventually a tissue specificexpression of this protein of interest.

Since it is possible to produce transgenic animals of the inventionusing a variety of different sequences, a general description will begiven of the production of transgenic animals by referring generally toexogenous genetic material. This general description can be adapted bythose skilled in the art in order to incorporate the DNA sequences intoanimals. For more details regarding the production of transgenicanimals, and specifically transgenic mice, it may be referred to Sandouet al. (1994) and also to U.S. Pat. No. 4,873,191, issued Oct. 10, 1989,U.S. Pat. No. 5,968,766, issued Dec. 16, 1997 and U.S. Pat. No.5,387,742, issued Feb. 28, 1995.

Transgenic animals of the present invention are produced by theapplication of procedures which result in an animal with a genome thatincorporates exogenous genetic material which is integrated into thegenome. The procedure involves obtaining the genetic material or aportion thereof, which encodes either a CIDE B coding sequence, a CIDE Bregulatory polynucleotide or a DNA sequence encoding an antisensepolynucleotide such as described in the present specification.

A recombinant polynucleotide of the invention is inserted into anembryonic or ES stem cell line. The insertion is made usingelectroporation. The cells subjected to electroporation are screened(e.g. Southern blot analysis) to find positive cells which haveintegrated the exogenous recombinant polynucleotide into their genome.An illustrative positive-negative selection procedure that may be usedaccording to the invention is described by Mansour et al. (1988). Then,the positive cells are isolated, cloned and injected into 3.5 days oldblastocysts from mice. The blastocysts are then inserted into a femalehost animal and allowed to grow to term. The offsprings of the femalehost are tested to determine which animals are transgenic e.g. includethe inserted exogenous DNA sequence and which are wild-type.

Thus, the present invention also concerns a transgenic animal containinga nucleic acid, a recombinant expression vector or a recombinant hostcell according to the invention.

Methods for Screening Substances Interacting with a CIDE B Polypeptide

For the purpose of the present invention, a ligand means a molecule,such as a protein, a peptide, an antibody or any synthetic chemicalcompound capable of binding to the CIDE B protein or one of itsfragments or variants or to modulate the expression of thepolynucleotide coding for CIDE B or a fragment or variant thereof.

In the ligand screening method according to the present invention, abiological sample or a defined molecule to be tested as a putativeligand of the CIDE B protein is brought into contact with the purifiedCIDE B protein, for example the purified recombinant CIDE B proteinproduced by a recombinant cell host as described hereinbefore, in orderto form a complex between the CIDE B protein and the putative ligandmolecule to be tested.

Another object of the present invention consists of methods and kits forthe screening of candidate substances that interact with a CIDE Bpolypeptide.

The present invention pertains to methods for screening substances ofinterest that interact with a CIDE B protein or one fragment or variantthereof. By their capacity to bind covalently or non-covalently to aCIDE B protein or to a fragment or variant thereof, these substances ormolecules may be advantageously used both in vitro and in vivo.

In vitro, said interacting molecules may be used as detection means inorder to identify the presence of a CIDE B protein in a sample,preferably a biological sample.

A method for the screening of a candidate substance interacting with aCIDE B polypeptide of the present invention comprises the followingsteps:

-   -   a) providing a polypeptide consisting of a CIDE B protein or a        fragment or a variant thereof;    -   b) obtaining a candidate substance;    -   c) bringing into contact said polypeptide with said candidate        substance;    -   d) detecting the complexes formed between said polypeptide and        said candidate substance.

In one embodiment of the screening method defined above, the complexesformed between the polypeptide and the candidate substance are furtherincubated in the presence of a polyclonal or a monoclonal antibody thatspecifically binds to the CIDE B protein or to said fragment or variantthereof.

Various candidate substances or molecules can be assayed for interactionwith a CIDE B polypeptide. These substances or molecules include,without being limited to, natural or synthetic organic compounds ormolecules of biological origin such as polypeptides. When the candidatesubstance or molecule consists of a polypeptide, this polypeptide may bethe resulting expression product of a phage clone belonging to aphage-based random peptide library, or alternatively the polypeptide maybe the resulting expression product of a cDNA library cloned in a vectorsuitable for performing a two-hybrid screening assay.

In another embodiment of the present screening method, increasingconcentrations of a monoclonal or polyclonal antibody directed against aCIDE B protein or a fragment or a variant thereof is reacted with theconsidered CIDE B protein or with a fragment or variant thereof,simultaneously or prior to the addition of the candidate substance ormolecule, when performing step c) of said method. By this technique, thedetection and optionally the quantification of the complexes formedbetween the CIDE B protein or the fragment or variant thereof and thesubstance or molecule to be screened allows the one skilled in the artto determine the affinity value of said substance or molecule for saidCIDE B protein or the fragment or variant thereof.

The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea CIDE B polypeptide or a fragment or a variant thereof and optionallymeans useful to detect the complex formed between the CIDE B polypeptideor its fragment or variant and the candidate substance. In a preferredembodiment the detection means consist in monoclonal or polyclonalantibodies directed against The CIDE B polypeptide or a fragment or avariant thereof.

1. Candidate Ligands Obtained from Random Peptide Libraries

In a particular embodiment of the screening method, the putative ligandis the expression product of a DNA insert contained in a phage vector(Parmley and Smith, 1988). Specifically, random peptide phages librariesare used. The random DNA inserts encode for peptides of 8 to 20 aminoacids in length (Oldenburg K. R. et al., 1992; Valadon P., et al., 1996;Lucas A. H., 1994; Westerink M. A. J., 1995; Castagnoli L. et al.,1991). According to this particular embodiment, the recombinant phagesexpressing a protein that binds to the immobilized CIDE B protein isretained and the complex formed between the CIDE B protein and therecombinant phage may be subsequently immunoprecipitated by a polyclonalor a monoclonal antibody directed against the CIDE B protein.

Once the ligand library in recombinant phages has been constructed, thephage population is brought into contact with the immobilized CIDE Bprotein. Then the preparation of complexes is washed in order to removethe non-specifically bound recombinant phages. The phages that bindspecifically to the CIDE B protein are then eluted by a buffer (acid pH)or immunoprecipitated by the monoclonal antibody produced by thehybridoma anti-CIDE B, and this phage population is subsequentlyamplified by an over-infection of bacteria (for example E. coli). Theselection step may be repeated several times, preferably 2–4 times, inorder to select the more specific recombinant phage clones. The laststep consists in characterizing the peptide produced by the selectedrecombinant phage clones either by expression in infected bacteria andisolation, expressing the phage insert in another host-vector system, orsequencing the insert contained in the selected recombinant phages.

2. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay

The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989), and relies upon the fusionof a bait protein to the DNA binding domain of the yeast Gal4 protein.This technique is also described in the U.S. Pat. No. 5,667,973 and theU.S. Pat. No. 5,283,173 (Fields et al.).

The general procedure of library screening by the two-hybrid assay maybe performed as described by Harper et al. (1993) or as described by Choet al. (1998) or also Fromont-Racine et al. (1997).

The bait protein or polypeptide consists of a CIDE B polypeptide or afragment or variant thereof.

More precisely, the nucleotide sequence encoding the CIDE B polypeptideor a fragment or variant thereof is fused to a polynucleotide encodingthe DNA binding domain of the GAL4 protein, the fused nucleotidesequence being inserted in a suitable expression vector, for examplepAS2 or pM3.

Then, a human cDNA library is constructed in a specially designedvector, such that the human cDNA insert is fused to a nucleotidesequence in the vector that encodes the transcriptional domain of theGAL4 protein. Preferably, the vector used is the pACT vector. Thepolypeptides encoded by the nucleotide inserts of the human cDNA libraryare termed “pray” polypeptides.

A third vector contains a detectable marker gene, such as betagalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative but nonlimiting example the two different yeast strains may be the followings:

-   -   Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12,        trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS        GAL-HIS3, cyh^(r));    -   Y187, the phenotype of which is (MATa gal4 gal80his3 trp1-901        ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet⁻), which is the        opposite mating type of Y190.

Briefly, 20 μg of pAS2/CIDE B and 20 μg of pACT-cDNA library areco-transformed into yeast strain Y190. The transformants are selectedfor growth on minimal media lacking histidine, leucine and tryptophan,but containing the histidine stnthesis inhibitor 3-AT (50 mM). Positivecolonies are screened for beta galactosidase by filter lift assay. Thedouble positive colonies (His⁺, beta-gal⁺) are then grown on plateslacking histidine, leucine, but containing tryptophan and cycloheximide(10 mg/ml) to select for loss of pAS2/CIDE B plasmids bu retention ofpACT-cDNA library plasmids. The resulting Y190 strains are mated withYl87 strains expressing CIDE B or non-related control proteins; such ascyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper etal. (1993) and by Bram et al. (1993), and screened for betagalactosidase by filter lift assay. Yeast clones that are beta gal-after mating with the control Gal4 fusions are considered falsepositives.

In another embodiment of the two-hybrid method according to theinvention, interaction between CIDE B or a fragment or variant thereofwith cellular proteins may be assessed using the Matchmaker Two HybridSystem 2 (Catalog No. K1604–1, Clontech).). As described in the manualaccompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604–1,Clontech), the disclosure of which is incorporated herein by reference,nucleic acids encoding the RBP-7 protein or a portion thereof, areinserted into an expression vector such that they are in frame with DNAencoding the DNA binding domain of the yeast transcriptional activatorGAL4. A desired cDNA, preferably human cDNA, is inserted into a secondexpression vector such that they are in frame with DNA encoding theactivation domain of GAL4. The two expression plasmids are transformedinto yeast and the yeast are plated on selection medium which selectsfor expression of selectable markers on each of the expression vectorsas well as GAL4 dependent expression of the HIS3 gene. Transformantscapable of growing on medium lacking histidine are screened for GAL4dependent lacZ expression. Those cells which are positive in both thehistidine selection and the lacZ assay contain interaction between CIDEB and the protein or peptide encoded by the initially selected cDNAinsert.

3. Candidate Ligands Obtained by Affinity Chromatography

Proteins or other molecules interacting with the CIDE B protein, or afragment thereof can also be found using affinity columns which containthe CIDE B protein, or a fragment thereof. The CIDE B protein, or afragment thereof, may be attached to the column using conventionaltechniques including chemical coupling to a suitable column matrix suchas agarose, Affi Gel®, or other matrices familiar to those of skill inart. In some embodiments of this method, the affinity column containschimeric proteins in which the CIDE B protein, or a fragment thereof, isfused to glutathion S transferase (GST). A mixture of cellular proteinsor pool of expressed proteins as described above is applied to theaffinity column. Proteins or other molecules interacting with the CIDE Bprotein, or a fragment thereof, attached to the column can then beisolated and analyzed on 2-D electrophoresis gel as described inRamunsen et al. (1997), the disclosure of which is incorporated byreference. Alternatively, the proteins retained on the affinity columncan be purified by electrophoresis based methods and sequenced. The samemethod can be used to isolate antibodies, to screen phage displayproducts, or to screen phage display human antibodies.

4. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with the CIDE B protein, or a fragment comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the amino acid positions 7–11, 18–29, 47,55–63, 70, 103–104, 111–115, 124, 134, 169–173, 181–185, and 203–219,can also be screened by using an Optical Biosensor as described inEdwards and Leatherbarrow (1997) and also in Szabo et al. (1995), thedisclosure of which is incorporated by reference. This technique permitsthe detection of interactions between molecules in real time, withoutthe need of labeled molecules. This technique is based on the surfaceplasmon resonance (SPR) phenomenon. Briefly, the candidate ligandmolecule to be tested is attached to a surface (such as a carboxymethyldextran matrix). A light beam is directed towards the side of thesurface that does not contain the sample to be tested and is reflectedby said surface. The SPR phenomenon causes a decrease in the intensityof the reflected light with a specific association of angle andwavelength. The binding of candidate ligand molecules cause a change inthe refraction index on the surface, which change is detected as achange in the SPR signal. For screening of candidate ligand molecules orsubstances that are able to interact with the CIDE B protein, or afragment thereof, the CIDE B protein, or a fragment thereof, isimmobilized onto a surface. This surface comprises one side of a cellthrough which flows the candidate molecule to be assayed. The binding ofthe candidate molecule on the CIDE B protein, or a fragment thereof, isdetected as a change of the SPR signal. The candidate molecules testedmay be proteins, peptides, carbohydrates, lipids, or small moleculesgenerated by combinatorial chemistry. This technique may also beperformed by immobilizing eukaryotic or prokaryotic cells or lipidvesicles exhibiting an endogenous or a recombinantly expressed CIDE Bprotein at their surface.

The main advantage of the method is that it allows the determination ofthe association rate between the CIDE B protein and moleculesinteracting with the CIDE B protein. It is thus possible to selectspecifically ligand molecules interacting with the CIDE B protein, or afragment thereof, through strong or conversely weak associationconstants.

Method for Screening Ligands that Modulate the Expression of the CIDE BGene

Another subject of the present invention is a method for screeningmolecules that modulate the expression of the CIDE B protein. Such ascreening method comprises the steps of:

-   -   a) cultivating a prokaryotic or an eukaryotic cell that has been        transfected with a nucleotide sequence encoding the CIDE B        protein or a variant or a fragment thereof, placed under the        control of its own promoter;    -   b) bringing into contact the cultivated cell with a molecule to        be tested;    -   c) quantifying the expression of the CIDE B protein or a variant        or a fragment thereof.

Using DNA recombination techniques well known by the one skill in theart, the CIDE B protein encoding DNA sequence is inserted into anexpression vector, downstream from its promoter sequence. As anillustrative example, the promoter sequence of the CIDE B gene iscontained in the 5′ regulatory region of CIDE B.

The quantification of the expression of the CIDE B protein may berealized either at the mRNA level or at the protein level. In the lattercase, polyclonal or monoclonal antibodies may be used to quantify theamounts of the CIDE B protein that have been produced, for example in anELISA or a RIA assay.

In a preferred embodiment, the quantification of the CIDE B mRNA isrealized by a quantitative PCR amplification of the cDNA obtained by areverse transcription of the total mRNA of the cultivated CIDEB-transfected host cell, using a pair of primers specific for CIDE B.

The present invention also concerns a method for screening substances ormolecules that are able to increase, or in contrast to decrease, thelevel of expression of the CIDE B gene. Such a method may allow the oneskilled in the art to select substances exerting a regulating effect onthe expression level of the CIDE B gene and which may be useful asactive ingredients included in pharmaceutical compositions for treatingpatients suffering from deficiencies in the regulation of expression ofthe CIDE B gene.

Thus, is also part of the present invention a method for the screeningof a candidate substance or molecule, said method comprising thefollowing steps:

-   -   a) providing a recombinant cell host containing a nucleic acid,        wherein said nucleic acid comprises a nucleotide sequence        selected from the group consisting of SEQ ID Nos 1 and 2;    -   b) obtaining a candidate substance, and    -   c) determining the ability of the candidate substance to        modulate the expression levels of the nucleotide sequences        selected from the group consisting of SEQ ID Nos 1 and 2.

The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea recombinant vector that allows the expression of a nucleotide sequenceselected from the group consisting of SEQ ID Nos 1 and 2 oralternatively a recombinant cell host containing such a recombinantvector.

In another embodiment of a method for screening of a candidate substanceor molecule that modulated the expression of the CIDE B gene, thismethod comprises the following steps:

-   -   a) providing a recombinant cell host containing a nucleic acid,        wherein said nucleic acid comprises the 5′ regulatory region of        CIDE B or a biologically active fragment or variant thereof        located upstream a polynucleotide encoding a detectable protein;    -   b) obtaining a candidate substance, and    -   c) determining the ability of the candidate substance to        modulate the expression levels of the polynucleotide encoding        the detectable protein.

Among the preferred polynucleotides encoding a detectable protein, theremay be cited polynucleotides encoding beta galactosidase, greenfluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT).

The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea recombinant vector comprising the 5′ regulatory region of CIDE B or abiologically active fragment or variant thereof located upstream andoperably linked to a polynucleotide encoding a detectable protein or theCIDE B protein or a fragment or a variant thereof.

For the design of suitable recombinant vectors useful for performing thescreening methods described above, it will be referred to the section ofthe present specification wherein the preferred recombinant vectors ofthe invention are detailed.

Expression levels and patterns of CIDE B may be analyzed by solutionhybridization with long probes as described in International PatentApplication No. WO 97/05277, the entire contents of which areincorporated herein by reference. Briefly, the CIDE B cDNA or the CIDE Bgenomic DNA described above, or fragments thereof, is inserted at acloning site immediately downstream of a bacteriophage (T3, T7 or SP6)RNA polymerase promoter to produce antisense RNA. Preferably, the CIDE Binsert comprises at least 100 or more consecutive nucleotides of thegenomic DNA sequence or the cDNA sequences, particularly thosecomprising at least one of SEQ ID Nos 15–18 or those encoding mutatedCIDE B. The plasmid is linearized and transcribed in the presence ofribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP andDIG-UTP). An excess of this doubly labeled RNA is hybridized in solutionwith mRNA isolated from cells or tissues of interest. The hybridizationsare performed under standard stringent conditions (40–50° C. for 16hours in an 80% formamide, 0.4 M NaCl buffer, pH 7–8). The unhybridizedprobe is removed by digestion with ribonucleases specific forsingle-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A). The presenceof the biotin-UTP modification enables capture of the hybrid on amicrotitration plate coated with streptavidin. The presence of the DIGmodification enables the hybrid to be detected and quantified by ELISAusing an anti-DIG antibody coupled to alkaline phosphatase.

Methods for Inhibiting the Expression of a CIDE B Gene

Other therapeutic compositions according to the present inventioncomprise advantageously an oligonucleotide fragment of the nucleicsequence of CIDE B as an antisense tool that inhibits the expression ofthe corresponding CIDE B gene. Preferred methods using antisensepolynucleotide according to the present invention are the proceduresdescribed by Sczakiel et al. (1995).

Preferably, the antisense tools are chosen among the polynucleotides(15–200 bp long) that are complementary to the 5′end of the CIDE B mRNA.In another embodiment, a combination of different antisensepolynucleotides complementary to different parts of the desired targetedgene are used.

Preferred antisense polynucleotides according to the present inventionare complementary to a sequence of the mRNAs of CIDE B that contains thetranslation initiation codon ATG.

The antisense nucleic acid molecules to be used in gene therapy may beeither DNA or RNA sequences. They comprise a nucleotide sequencecomplementary to the targeted sequence of the CIDE B genomic DNA, thesequence of which can be determined using one of the detection methodsof the present invention. In a preferred embodiment, the antisenseoligonucleotide are able to hybridize with at least one of the splicingsites of the targeted CIDE B gene, or with the 3′UTR of the 5′UTR. Theantisense nucleic acids should have a length and melting temperaturesufficient to permit formation of an intracellular duplex havingsufficient stability to inhibit the expression of the CIDE B mRNA in theduplex. Strategies for designing antisense nucleic acids suitable foruse in gene therapy are disclosed in Green et al., (1986) and Izant andWeintraub, (1984).

In some strategies, antisense molecules are obtained by reversing theorientation of the CIDE B coding region with respect to a promoter so asto transcribe the opposite strand from that which is normallytranscribed in the cell. The antisense molecules may be transcribedusing in vitro transcription systems such as those which employ T7 orSP6 polymerase to generate the transcript. Another approach involvestranscription of CIDE B antisense nucleic acids in vivo by operablylinking DNA containing the antisense sequence to a promoter in asuitable expression vector.

Alternatively, suitable antisense strategies are those described byRossi et al. (1991), in the International Applications Nos. WO 94/23026,WO 95/04141, WO 92/18522 and in the European Patent Application No. EP 0572 287 A2

An alternative to the antisense technology that is used according to thepresent invention consists in using ribozymes that will bind to a targetsequence via their complementary polynucleotide tail and that willcleave the corresponding RNA by hydrolyzing its target site (namely<<hammerhead ribozymes>>. Briefly, the simplified cycle of a hammerheadribozyme consists of (1) sequence specific binding to the target RNA viacomplementary antisense sequences; (2) site-specific hydrolysis of thecleavable motif of the target strand; and (3) release of cleavageproducts, which gives rise to another catalytic cycle. Indeed, the useof long-chain antisense polynucleotide (at least 30 bases long) orribozymes with long antisense arms are advantageous. A preferreddelivery system for antisense ribozyme is achieved by covalently linkingthese antisense ribozymes to lipophilic groups or to use liposomes as aconvenient vector. Preferred antisense ribozymes according to thepresent invention are prepared as described by Sczakiel et al. (1995).

Throughout this application, various publications, patents and publishedpatent applications are cited. The disclosures of these publications,patents and published patent specification referenced in thisapplication are hereby incorporated by reference into the presentdisclosure to more fully describe the sate of the art to which thisinvention pertains.

EXAMPLES Example 1 Identification of Biallelic Markers—DNA Extraction

Donors were unrelated and healthy. They presented a sufficient diversityfor being representative of a French heterogeneous population. The DNAfrom 100 individuals was extracted and tested for the detection of thebiallelic markers.

30 ml of peripheral venous blood were taken from each donor in thepresence of EDTA. Cells (pellet) were collected after centrifugation for10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50 mlfinal volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solutionwas centrifuged (10 minutes, 2000 rpm) as many times as necessary toeliminate the residual red cells present in the supernatant, afterresuspension of the pellet in the lysis solution.

The pellet of white cells was lysed overnight at 42° C. with 3.7 ml oflysis solution composed of:

-   -   3 ml TE 10–2 (Tris-HCl 10 mM, EDTA 2 mM)NaCl 0 4 M    -   200 μl SDS 10%    -   500 μl K-proteinase (2 mg K-proteinase in TE 10–2/NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) wasadded. After vigorous agitation, the solution was centrifuged for 20minutes at 10000 rpm.

For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were addedto the previous supernatant, and the solution was centrifuged for 30minutes at 2000 rpm. The DNA solution was rinsed three times with 70%ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm.The pellet was dried at 37° C., and resuspended in 1 ml TE 10–1 or 1 mlwater. The DNA concentration was evaluated by measuring the OD at 260 nm(1 unit OD=50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260/OD280 ratio was determined. Only DNA preparations having a OD 260/OD 280ratio between 1.8 and 2 were used in the subsequent examples describedbelow.

The pool was constituted by mixing equivalent quantities of DNA fromeach individual.

Example 2 Identification of Biallelic Markers: Amplification of GenomicDNA by PCR

The amplification of specific genomic sequences of the DNA samples ofexample 1 was carried out on the pool of DNA obtained previously. Inaddition, 50 individual samples were similarly amplified.

PCR assays were performed using the following protocol:

Final volume 25 μl DNA 2 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer(each) 2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer(10x = 0.1 M TrisHCl pH 8.3 0.5 M KCl) 1×

Each pair of first primers was designed using the sequence informationof the CIDE B gene disclosed herein and the OSP software (Hillier &Green, 1991). This first pair of primers was about 20 nucleotides inlength and had the sequences disclosed in Table 1 in the columns labeledPU and RP.

TABLE 1 Position Complementary range of the Position range of positionrange of amplicon in Primer amplification primer Primer amplificationprimer Amplicon SEQ ID 1 name in SEQ ID No 1 name in SEQ ID No 2 12–736704 7169 B(12–73) 6704 6723 C(12–73) 7152 7169 12–74 9538 9988 B(12–74)9538 9557 C(12–74) 9970 9988

Preferably, the primers contained a common oligonucleotide tail upstreamof the specific bases targeted for amplification which was useful forsequencing.

Primers PU contain the following additional PU 5′ sequence:TGTAAAACGACGGCCAGT; primers RP contain the following RP 5′ sequence:CAGGAAACAGCTATGACC. The primer containing the additional PU 5′ sequenceis listed in SEQ ID No 4. The primer containing the additional RP 5′sequence is listed in SEQ ID No 5.

The synthesis of these primers was performed following thephosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

DNA amplification was performed on a Genius II thermocycler. Afterheating at 95° C. for 10 min, 40 cycles were performed. Each cyclecomprised: 30 sec at 95° C., 54° C. for 1 min, and 30 sec at 72° C. Forfinal elongation, 10 min at 72° C. ended the amplification. Thequantities of the amplification products obtained were determined on96-well microtiter plates, using a fluorometer and Picogreen asintercalant agent (Molecular Probes).

Example 3 Identification of Biallelic Markers—Sequencing of AmplifiedGenomic DNA and Identification of Polymorphisms

The sequencing of the amplified DNA obtained in example 2 was carriedout on ABI 377 sequencers. The sequences of the amplification productswere determined using automated dideoxy terminator sequencing reactionswith a dye terminator cycle sequencing protocol. The products of thesequencing reactions were run on sequencing gels and the sequences weredetermined using gel image analysis (ABI Prism DNA Sequencing Analysissoftware (2.1.2 version)).

The sequence data were further evaluated to detect the presence ofbiallelic markers within the amplified fragments. The polymorphismsearch was based on the presence of superimposed peaks in theelectrophoresis pattern resulting from different bases occurring at thesame position as described previously.

In the 2 fragments of amplification, 2 biallelic markers were detected.The localization of these biallelic markers are as shown in Table 2.

TABLE 2 Localization Marker in CIDE Polymorphism BM position in AmpliconName B gene allele1 allele2 SEQ ID No 1 12-73 12-73-49 3′ regulatory C T7123 region 12-74 12-74-38 3′ regulatory C T 9574 region

TABLE 3 Position range of Marker Name probes in SEQ ID No 1 Probes12-73-49 7100 7146 P(12-73-49) 12-74-38 9551 9597 P(12-74-38)

Example 4 Validation of the Polymorphisms through Microsequencing

The biallelic markers identified in example 3 were further confirmed andtheir respective frequencies were determined through microsequencing.Microsequencing was carried out for each individual DNA sample describedin Example 1.

Amplification from genomic DNA of individuals was performed by PCR asdescribed above for the detection of the biallelic markers with the sameset of PCR primers (Table 1).

The preferred primers used in microsequencing were about 19 nucleotidesin length and hybridized just upstream of the considered polymorphicbase. According to the invention, the primers used in microsequencingare detailed in Table 4.

TABLE 4 Position range of Complementary position microsequencing rangeof Marker primer mis 1 in microsequencing primer Name Mis. 1 SEQ ID No 1Mis. 2 mis. 2 in SEQ ID No 1 12-73-49 D(12-73-49) 7104 7122 E(12-73-49)7124 7142 12-74-38 D(12-74-38) 9555 9573 E(12-74-38) 9575 9593

Mis 1 and Mis 2 respectively refer to microsequencing primers whichhybridized with the non-coding strand of the CIDE B gene or with thecoding strand of the CIDE B gene.

The microsequencing reaction was performed as follows:

After purification of the amplification products, the microsequencingreaction mixture was prepared by adding, in a 20 μl final volume: 10pmol microsequencing oligonucleotide, 1 U Thermosequenase (AmershamE79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mMMgCl₂), and the two appropriate fluorescent ddNTPs (Perkin Elmer, DyeTerminator Set 401095) complementary to the nucleotides at thepolymorphic site of each biallelic marker tested, following themanufacturer's recommendations. After 4 minutes at 94° C., 20 PCR cyclesof 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. were carriedout in a Tetrad PTC-225 (MJ Research). The unincorporated dyeterminators were then removed by ethanol precipitation. Samples werefinally resuspended in formamide-EDTA loading buffer and heated for 2min at 95° C. before being loaded on a polyacrylamide sequencing gel.The data were collected by an ABI PRISM 377 DNA sequencer and processedusing the GENESCAN software (Perkin Elmer).

Following gel analysis, data were automatically processed with softwarethat allows the determination of the alleles of biallelic markerspresent in each amplified fragment.

The software evaluates such factors as whether the intensities of thesignals resulting from the above microsequencing procedures are weak,normal, or saturated, or whether the signals are ambiguous. In addition,the software identifies significant peaks (according to shape and heightcriteria). Among the significant peaks, peaks corresponding to thetargeted site are identified based on their position. When twosignificant peaks are detected for the same position, each sample iscategorized classification as homozygous or heterozygous type based onthe height ratio.

Example 5 Preparation of Antibody Compositions to the CIDE B Protein

Substantially pure protein or polypeptide is isolated from transfectedor transformed cells containing an expression vector encoding the CIDE Bprotein or a portion thereof. The concentration of protein in the finalpreparation is adjusted, for example, by concentration on an Amiconfilter device, to the level of a few micrograms/ml. Monoclonal orpolyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes in the CIDE B protein or a portionthereof can be prepared from murine hybridomas according to theclassical method of Kohler, G. and Milstein, C., (1975) or derivativemethods thereof. Also see Harlow, E., and D. Lane. 1988.

Briefly, a mouse is repetitively inoculated with a few micrograms of theCIDE B protein or a portion thereof over a period of a few weeks. Themouse is then sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and the excess unfused cells destroyed bygrowth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, (1980), and derivative methods thereof. Selected positiveclones can be expanded and their monoclonal antibody product harvestedfor use. Detailed procedures for monoclonal antibody production aredescribed in Davis, L. et al. Basic Methods in Molecular BiologyElsevier, New York. Section 21-2.

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes inthe CIDE B protein or a portion thereof can be prepared by immunizingsuitable non-human animal with the CIDE B protein or a portion thereof,which can be unmodified or modified to enhance immmunogenicity. Asuitable non-human animal is preferably a non-human mammal is selected,usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crudepreparation which has been enriched for CIDE B concentration can be usedto generate antibodies. Such proteins, fragments or preparations areintroduced into the non-human mammal in the presence of an appropriateadjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in theart. In addition the protein, fragment or preparation can be pretreatedwith an agent which will increase antigenicity, such agents are known inthe art and include, for example, methylated bovine serum albumin(mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, andkeyhole limpet hemocyanin (KLH). Serum from the immunized animal iscollected, treated and tested according to known procedures. If theserum contains polyclonal antibodies to undesired epitopes, thepolyclonal antibodies can be purified by immunoaffinity chromatography.

Effective polyclonal antibody production is affected by many factorsrelated both to the antigen and the host species. Also, host animalsvary in response to site of inoculations and dose, with both inadequateor excessive doses of antigen resulting in low titer antisera. Smalldoses (ng level) of antigen administered at multiple intradermal sitesappears to be most reliable. Techniques for producing and processingpolyclonal antisera are known in the art, see for example, Mayer andWalker (1987). An effective immunization protocol for rabbits can befound in Vaitukaitis, J. et al. (1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Afinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,(1980).

Antibody preparations prepared according to either the monoclonal or thepolyclonal protocol are useful in quantitative immunoassays whichdetermine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample. The antibodiesmay also be used in therapeutic compositions for killing cellsexpressing the protein or reducing the levels of the protein in thebody.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein by the one skilled in the art without departing from the spiritand scope of the invention.

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SEQUENCE LISTING FREE TEXT

The following free text appears in the accompanying Sequence Listing:

-   5′ regulatory region-   3′ regulatory region-   polymorphic base or-   complement-   probe-   homology with 5′ EST in ref-   sequencing oligonucleotide Primer

1. An isolated, purified, or recombinant polypeptide comprising SEQ IDNO:
 3. 2. An isolated, purified, or recombinant polypeptide encoded bythe polynucleotide sequence of SEQ ID NO:
 2. 3. An isolated, purified,or recombinant polypeptide encoded by a polynucleotide sequence selectedfrom the group consisting of: a) nucleotide positions 2803–2922 of SEQID NO: 1; b) nucleotide positions 3225–3369 of SEQ ID NO: 1; c)nucleotide positions 4603–4793 of SEQ ID NO: 1; d) nucleotide positions1–2802 of SEQ ID NO: 1; e) nucleotide positions 80–739 of SEQ ID NO: 2;f) a nucleotide sequence encoding a contiguous span of at least 35 aminoacids of SEQ ID NO: 3; g) a nucleotide sequence encoding a contiguousspan of at least 40 amino acids of SEQ ID NO: 3; h) a nucleotidesequence encoding a contiguous span of at least 50 amino acids of SEQ IDNO): 3; i) a nucleotide sequence encoding a contiguous span of at least100 amino acids of SEQ ID NO): 3; j) a nucleotide sequence encoding acontiguous span of at least 150 amino acids of SEQ ID NO: 3; k) anucleotide sequence encoding a contiguous span of at least 200 aminoacids of SEQ ID NO: 3; l) a nucleotide sequence encoding amino acidpositions 1–29 of SEQ ID NO: 3; m) a nucleotide sequence encoding aminoacid positions 47–70 of SEQ ID NO: 3; and n) a nucleotide sequenceencoding amino acid positions 169–185 of SEQ ID NO:
 3. 4. The isolated,purified, or recombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is a).
 5. The isolated, purified, or recombinantpolypeptide according to claim 3, wherein said polynucleotide sequenceis b).
 6. The isolated, purified, or recombinant polypeptide accordingto claim 3, wherein said polynucleotide sequence is c).
 7. The isolated,purified, or recombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is d).
 8. The isolated, purified, or recombinantpolypeptide according to claim 3, wherein said polynucleotide sequenceis e).
 9. The isolated, purified, or recombinant polypeptide accordingto claim 3, wherein said polynucleotide sequence is f).
 10. Theisolated, purified, or recombinant polypeptide according to claim 3,wherein said polynucleotide sequence is g).
 11. The isolated, purified,or recombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is h).
 12. The isolated, purified, orrecombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is i).
 13. The isolated, purified, orrecombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is j).
 14. The isolated, purified, orrecombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is k).
 15. The isolated, purified, orrecombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is l).
 16. The isolated, purified, orrecombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is m).
 17. The isolated, purified, orrecombinant polypeptide according to claim 3, wherein saidpolynucleotide sequence is n).